Lihui Zou

Extrachromosomal circular DNA (eccDNA) is a class of circular DNA molecules that originate from and are independent of conventional chromosomes. The high specificity and stability of the circular structure of eccDNA provide new clues for its research as a disease biomarker. The recent rapid development of long-read sequencing and eccDNA identification algorithms has greatly expanded our understanding of eccDNA properties. In this review, we introduce the molecular characteristics, biological origin, and advances in detection technology of eccDNA, and mainly summarize the clinical implications of eccDNA in cancer diagnosis and prognosis, drug resistance monitoring, prenatal testing, and immune-related disease, providing support for further exploring new clinical applications of eccDNA.

The purpose of this study was to explore the composition and function of immune cell subsets at the single-cell level in the thymus and peripheral blood of patients with myasthenia gravis (MG). A total of 9701 and 23 846 cells, respectively, originated from the peripheral blood and thymus samples of two MG patients, and 6930 cells from the peripheral blood of two gender- and age-matched healthy controls (HCs) were selected for single-cell RNA-sequencing. Uniform manifold approximation and projection (UMAP), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Monocle3, and Velcyto were performed to analyze the composition, molecular and functional properties, and developmental trajectory of immune cell subsets. Four major cell populations of T cells, B cells, myeloid cells, and natural killer cells were identified, as well as their 15 cell subpopulations. An absolute predominance of T cells was found in the thymus and peripheral blood of MG patients, and the proportions of memory B cells in both plasma and thymus showed an increasing trend while the number of naïve B cells demonstrated a decreasing trend in MG patients compared with HCs. Besides, the monocytes in the peripheral blood of MG patients had the strongest interactions with other cells. Furthermore, CXCL, GAS, and CD30 signaling pathways were more enriched within MG peripheral blood. Our research clarifies the cellular heterogeneity in the pathogenesis of MG and characterizes the immune microenvironment of thymic tissues in MG patients.

Extrachromosomal circular DNA (eccDNA) is an emerging class of genetic material that exists outside of the chromosomal genome. These circular DNA molecules are gaining increasing attention as important biomarkers in various cancers because of their roles in gene amplification, genetic heterogeneity, and drug resistance. In this review, we explore in depth the impacts of eccDNAs on cancer biology, their potential to predict treatment sensitivity and resistance, and their involvement in the development of new anticancer therapies. eccDNAs can be used as biomarkers for various tumor types to help diagnose and predict prognosis.

The distribution and the function of T lymphocyte subsets in pleural effusion (PE) and peripheral blood (PB) in tuberculous pleural effusion (TPE) patients remain unclear. In this study, we aimed to explore the expression patterns, regulatory mechanisms, and functions of T lymphocyte subsets in TPE patients, especially the distribution of T lymphocyte subsets at the single cell level. The CD3⁺ T cells were isolated from PE and PB of four TPE patients for single‐cell RNA sequencing (scRNA‐seq) to screen T cell subsets. T‐SNE projection, Gene Set Variation Analysis (GSVA), and pseudotime analysis were performed to analyze the composition, molecular and functional properties, and developmental trajectories of T cell subsets. Finally, ELISA was carried out to identify the cytokines secreted by PE and PB. We found that CD4⁺CD8⁻ T lymphocytes (Th1, Th2, and FOXP3⁺ Treg cells) were preferentially enriched in PE. The proportion of exhausted CD4⁻CD8⁺ cells in PE was higher than that in PB, while the proportion of initial and effector CD4⁻CD8⁺ cells was quite the reverse. We also found a large number of unexpected double positive (DP) cells in PE and PB, among which the proportion of CD4⁺CD8⁺‐C10‐CCL3 cells was the most different between PE and PB. Meanwhile, CD4⁺CD8⁺‐C10‐CCL3 was the group with the largest number of interactions with other groups. CD4⁻CD8⁻ cells were mainly found in PE and may be involved in the immunomodulatory effect of PE. Furthermore, the concentrations of cytokines secreted by Th1, Th2, and Treg in PE were higher than those in PB. Our study is helpful to understand the distribution pattern and dynamic changes of T cells in PE and PB of TPE patients and further understand that the functional status and regulation of T cells will be crucial for the successful development of TPE immunotherapy.

Purpose Recombinant human B-type natriuretic peptide (rhBNP) has been extensively proven to be an effective mean of heart failure (HF) therapy, but its clinical application is limited by its very short half-life. This study aims to combine in vitro transcribed mRNA (IVT mRNA) and fusion protein technology to develop a rhBNP-Fc mRNA drug with long half-life, high efficiency and few side effects to treat HF. Methods The rhBNP-Fc fusion mRNA with IgG4-Fc sequence was produced by IVT technology. rhBNP-Fc mRNA was transfected into HEK293T cells to examine the expression in vitro. rhBNP-Fc mRNA encapsulated in LNP was injected into normal mice to detect the translation efficiency, half-life and negative effects in vivo. Finally, it was injected into doxorubicin-induced HF mice to screen the cardiac protective effect. Results The rhBNP-Fc fusion mRNA extended the half-life of rhBNP, showing sustained expression in cell line for at least one day. rhBNP-Fc mRNA translation showed dose-dependent levels, and was still detectable 5 d after injection in vivo. In the HF mouse model, a single administration of rhBNP-Fc mRNA-LNP improved cardiac function, including improving heart ejection and reducing HF biomarkers expression. Additionally, rhBNP-Fc mRNA-LNP treatment mitigated myocardial damage, normalized cardiomyocyte structure, and reduced the levels of pro-inflammatory cytokines. Conclusion The rhBNP-Fc mRNA has the potential to serve as an alternative to traditional protein therapies, thereby reducing clinical dosages, injection frequencies, and treatment costs. Our findings offer new insights into the development and application of mRNA drugs, emphasizing their therapeutic potential in long-acting drugs.

Due to the diversity of the immune repertoire (IR), reconstructing full-length IR using traditional short-read sequencing has proven challenging. A full-length IR sequencing (FLIRseq) work flow was developed with linear rolling circle amplification and nanopore sequencing. Its accuracy and quantification ability were verified by plasmid mixtures and commercial B-cell receptor/T-cell receptor sequencing (BCR/TCR-seq) based on short reads. IRs in tissues and the peripheral blood from 8 patients with acute lymphoblastic leukemia, 3 patients with allergic diseases, 4 patients with psoriasis, and 5 patients with prostate cancer were analyzed using FLIRseq. FLIRseq reads had lower mismatch rates and gap rates, and higher identify rates than nanopore reads (all P < 2.2 × ⁻¹⁶). The relative quantification of components by FLIRseq was consistent with the actual quantification (P > 0.05). FLIRseq had superiority over BCR/TCR-seq, providing the long complementarity-determining region 3, B-cell isotype, and the rarely used V gene sequence. FLIRseq observed an increase in clonotype diversity (P < 0.05) and a decrease in the percentage of abnormal BCRs/TCRs in patients with leukemia in remission. For patients with allergic diseases or psoriasis, FLIRseq provided direct insights into V(D)J recombination and specific immunoglobulin classes. Compared with that in prostate cancer tissues, the full-length V segment of the biased T-cell receptor β chain from lymphocytes in psoriatic tissues showed a more consistent AlphaFold2-predicted protein structure (P < 0.05). FLIRseq enables unbiased and comprehensive analyses of direct V(D)J recombination and immunoglobulin classes, thereby contributing to characterizing pathogenic mechanisms, monitoring minimal residual disease, and customizing adoptive cell therapy.

The surge of genome sequencing data has underlined substantial genetic variants of uncertain significance (VUS). The decryption of VUS discovered by sequencing poses a major challenge in the post-sequencing era. Although experimental assays have progressed in classifying VUS, only a tiny fraction of the human genes have been explored experimentally. Thus, it is urgently needed to generate state-of-the-art functional predictors of VUS in silico. Artificial intelligence (AI) is an invaluable tool to assist in the identification of VUS with high efficiency and accuracy. An increasing number of studies indicate that AI has brought an exciting acceleration in the interpretation of VUS, and our group has already used AI to develop protein structure-based prediction models. In this review, we provide an overview of the previous research on AI-based prediction of missense variants, and elucidate the challenges and opportunities for protein structure-based variant prediction in the post-sequencing era.

Background: Small extrachromosomal circular DNAs (eccDNAs) have the potential to be cancer biomarkers. However, the formation mechanisms and functions of small eccDNAs selected in carcinogenesis are not clear, and whether the small eccDNA profile in the plasma of cancer patients represents that in cancer tissues remains to be elucidated.Methods: A novel sequencing workflow based on the nanopore sequencing platform was used to sequence naturally existing full‐length small eccDNAs in tissues and plasma collected from 25 cancer patients (including prostate cancer, hepatocellular carcinoma and colorectal cancer), and from an independent validation cohort (including 7 cancer plasma and 14 healthy plasma).Results: Compared with those in non‐cancer tissues, small eccDNAs detected in cancer tissues had a significantly larger number and size (P = 0.040 and 2.2e‐16, respectively), along with more even distribution and different formation mechanisms. Although small eccDNAs had different general characteristics and genomic annotation between cancer tissues and the paired plasma, they had similar formation mechanisms and cancer‐related functions. Small eccDNAs originated from some specific genes had great multi‐cancer diagnostic value in tissues (AUC ≥ 0.8) and plasma (AUC > 0.9), especially increasing the accuracy of multi‐cancer prediction of CEA/CA19‐9 levels. The high multi‐cancer diagnostic value of small eccDNAs originated from ALK&ETV6 could be extrapolated from tissues (AUC = 0.804) to plasma and showed high positive predictive value (100%) and negative predictive value (82.35%) in a validation cohort.Conclusions: As independent and stable circular DNA molecules, small eccDNAs in both tissues and plasma can be used as ideal biomarkers for cost‐effective multi‐cancer diagnosis and monitoring.

The heart is a muscular organ that pumps blood throughout the body and is one of the most vital organs in human body. While cardiomyocytes are essential for maintaining the normal function of the heart, a variety of cardiovascular diseases such as coronary artery occlusion, arrhythmia, and myocarditis can lead to cardiomyocyte death, resulting in deterioration of heart function. The adult mammalian heart is incapable of regenerating sufficient cardiomyocytes following cardiac injuries, eventually leading to heart failure and death. Cardiac macrophages are ubiquitously distributed in the healthy heart and accumulated at the site of injury. Macrophages play essential roles in regulating homeostasis and proliferation of cardiomyocyte, promoting electrical conduction, and removing dead cardiomyocytes and debris through direct and indirect cell–cell crosstalk. In this review, we summarize the latest insights into the role of macrophages in maintaining cardiac homeostasis and the macrophage-cardiomyocyte crosstalk in both healthy and injured scenarios.

Classic strategies for circular RNA (circRNA) preparation always introduce large numbers of linear transcripts or extra nucleotides to the circularized product. In this study, we aimed to develop an efficient system for circRNA preparation based on a self-splicing ribozyme derived from an optimized Tetrahymena thermophila group Ⅰ intron. The target RNA sequence was inserted downstream of the ribozyme and a complementary antisense region was added upstream of the ribozyme to assist cyclization. Then, we compared the circularization efficiency of ribozyme or flanking intronic complementary sequence (ICS)-mediated methods through the DNMT1, CDR1as, FOXO3, and HIPK3 genes and found that the efficiency of our system was remarkably higher than that of flanking ICS-mediated method. Consequently, the circularized products mediated by ribozyme are not introduced with additional nucleotides. Meanwhile, the overexpressed circFOXO3 maintained its biological functions in regulating cell proliferation, migration, and apoptosis. Finally, a ribozyme-based circular mRNA expression system was demonstrated with a split green fluorescent protein (GFP) using an optimized Coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) sequence, and this system achieved successful translation of circularized mRNA. Therefore, this novel, convenient, and rapid engineering RNA circularization system can be applied for the functional study and large-scale preparation of circular RNA in the future.