Search Results (1,645)

Background: Zebrafish-based drug discovery systems provide significant advantages over mammalian models for high-throughput in vivo screening. Among these, the NF-κB eGFP reporter system significantly enhances drug discovery in zebrafish by enabling real-time, high-resolution monitoring of pathway activity in live organisms, thereby streamlining mechanistic studies and high-throughput screening. Methods: We developed a novel AI (Quantifish and Orange software)-based zebrafish precision individualized 96-well ZF plates (0–7 dpf) and individualized MT tanks (8 dpf–4 mpf) protocol for the transposon-mediated transgenesis of the NFκB eGFP reporter system. Results: One-cell stage embryos were administered NFκB reporter construct and Tol2 transposase mRNA via microinjection and transferred to separate wells of a 96-well ZF plate. Bright-field and fluorescence images of each well were captured at 5 dpf in the F0, F1, and F2 generations using the automated confocal high-content imager CQ1. The Quantifish software was used for the automated detection and segmentation of zebrafish larval fluorescence intensity in specific regions of interest. Quantitative data on the fluorescence intensity and distribution patterns were measured in Quantifish, and advanced statistical and machine learning methods were applied using Orange. Imaging data with eGFP expression results were assessed to evaluate the efficiency of the transgenic protocol. Discussion: This AI-enhanced precision protocol allows for high-throughput screening and quantitative analysis of NFκB reporter transgenesis in zebrafish, enabling the efficient identification and characterization of stable transgenic lines that exhibit tissue-specific expression of the NF-κB reporter, such as lines with induced expression restricted to the retina following LPS stimulation. This approach streamlines the evaluation of regulatory elements, enhances data consistency, and reduces animal use, making it a valuable tool for zebrafish drug discovery. Full article

Monkeypox virus (MPXV) experienced an unprecedented global outbreak in 2022, characterized by a significant departure from historical patterns: a rapid spread of the epidemic to more than 110 non-traditional endemic countries, with more than 90,000 confirmed cases; a fundamental shift in the mode of transmission, with human-to-human transmission (especially among men who have sex with men (MSM)) becoming the dominant route (95.2%); and genetic sequencing revealing a key adaptive mutation in a novel evolutionary branch (Clade IIb) that triggered the outbreak. These features highlight the significant evolution of MPXV in terms of host adaptation, transmission efficiency, and immune escape ability. The aim of this paper is to provide insights into the viral adaptive evolutionary mechanisms driving this global outbreak, with a particular focus on the role of immune escape (e.g., novel mechanisms of M2 proteins targeting the T cell co-stimulatory pathway) in enhancing viral transmission and pathogenicity. At the same time, we systematically evaluate the cross-protective efficacy and limitations of existing vaccines (ACAM2000, JYNNEOS, and LC16), as well as recent advances in novel vaccine platforms, especially mRNA vaccines, in inducing superior immune responses. The study further reveals the constraints to outbreak control posed by grossly unequal global vaccine distribution (e.g., less than 10% coverage in high-burden regions such as Africa) and explores the urgency of optimizing stratified vaccination strategies and facilitating technology transfer to promote equitable access. The core of this paper is to elucidate the dynamic game between viral evolution and prevention and control strategies (especially vaccines). The key to addressing the long-term epidemiological challenges of MPXV in the future lies in continuously strengthening global surveillance of viral evolution (early warning of highly transmissible/pathogenic variants), accelerating the development of next-generation vaccines based on new mechanisms and platforms (e.g., multivalent mRNAs), and resolving the vaccine accessibility gap through global collaboration to build an integrated defense system of “Surveillance, Research and Development, and Equitable Vaccination,” through global collaboration to address the vaccine accessibility gap. Full article

Extracellular vesicles (EVs) are mediators of intercellular communication and serve as promising tools for drug discovery and targeted therapies. These lipid bilayer-bound nanovesicles facilitate the transfer of functional proteins, RNAs, lipids, and other biomolecules between cells, thereby influencing various physiological and pathological processes. This review outlines the molecular mechanisms governing EV biogenesis and cargo sorting, emphasizing the role of key regulatory proteins in modulating selective protein packaging. We explore the critical involvement of EVs in various disease microenvironments, including cancer progression, neurodegeneration, and immunological modulation. Their ability to cross biological barriers and deliver bioactive cargo makes them desirable candidates for precise drug delivery systems, especially in neurological and oncological disorders. Moreover, this review highlights advances in engineering EVs for the delivery of RNA therapeutics, CRISPR-Cas systems, and targeted small molecules. The utility of EVs as diagnostic tools in liquid biopsies and their integration into personalized medicine and companion diagnostics are also discussed. Patient-derived EVs offer dynamic insights into disease states and enable real-time treatment stratification. Despite their potential, challenges such as scalable isolation, cargo heterogeneity, and regulatory ambiguity remain significant hurdles. Recent studies have reported novel pharmacological approaches targeting EV biogenesis, secretion, and uptake pathways, with emerging regulators showing promise as drug targets for modulating EV cargo. Future directions include the standardization of EV analytics, scalable biomanufacturing, and the classification of EV-based therapeutics under evolving regulatory frameworks. This review emphasizes the multifaceted roles of EVs and their transformative potential as therapeutic platforms and biomarker reservoirs in next-generation precision medicine. Full article

The development of low-background, facile, and robust fluorescent nanoprobes for imaging and monitoring of intracellular mRNA changes remains a great challenge. Taking advantage of the high fluorescence quenching efficiency of core-shell gold@polydopamine (Au@PDA) nanocomposites and Ca²⁺-promoting DNA adsorption stability, a simple and universal bioconjugate strategy was designed to a construct fluorescent nanoprobe for highly efficient tumor-related mRNA imaging. The fluorescence of Cy5-labeled DNA was quenched up to 92.38% by the AuNP and PDA via nanometal surface energy transfer (NSET) and photoinduced electron transfer (PET), respectively. TK1 mRNA, a biomarker of tumor growth, initiates hybridization and results in fluorescence recovery, which built the foundation for identifying the expression level changes in living cells. More importantly, three kinds of tumor-related mRNA (TK1 mRNA, GalNAc-T mRNA, and C-myc mRNA) can be detected simultaneously with different fluorophore-modified recognition sequences, which can avoid false positive signals and improve the reliability of cancer diagnostic, holding great promise for cancer diagnosis, prognosis, and therapy. Full article

Background/Objectives: Transfer RNA-derived fragments (tRFs) are small non-coding RNAs increasingly implicated in gene regulation and disease, yet their target specificity and disease relevance remain poorly understood. This is an exploratory study that investigates the phenomenon of identical tRF sequences reported in distinct disease contexts and evaluates the consistency between experimental findings and predictions from both target-based and abundance-based tRF databases. Methods: Five tRFs with identical sequences across at least two peer-reviewed disease studies were selected from a recent systematic review. Their validated targets and disease associations were extracted from the literature. Motifs and predicted targets were cross-referenced using three target-oriented databases: tatDB, tRFTar, and tsRFun. In parallel, the abundance enrichment of cancer-associated tRFs was assessed in OncotRF and MINTbase using TCGA-based abundance data. Results: Among the five tRFs, only LeuAAG-001-N-3p-68-85 showed complete alignment between experimental data and both tatDB and tRFTar predictions. Most of the other four displayed at least partial overlaps in motif/binding regions with some of validated targets. tRF abundance data from MINTbase and OncotRF showed inconsistent enrichment, with only AlaAGC-002-N-3p-58-75 exhibiting concordance with its experimentally validated cancer type. Most functionally relevant tRFs were not strongly represented in abundance-only databases. Conclusions: Given the limited number of tRFs analyzed, this study serves primarily as a pilot analysis designed to generate hypotheses and guide future in-depth research, rather than offering comprehensive conclusions. We did, however, illustrate how the analysis of tRFs can benefit from utilizing currently available databases. Target-based databases more closely reflected experimental evidence for mechanistic details when a tRF or a motif match is found. Yet all database types are incomplete, including the abundance-focused tools, which often fail to capture disease-specific regulatory roles of tRFs. These findings underscore the importance of using integrated data sources for tRF annotation. As a pilot analysis, the study provides insights into how identical tRF sequences might function differently across disease contexts, highlighting areas for further investigation while pointing out the limitations of relying on expression data alone to infer functional relevance. Full article

Clonorchis sinensis, a parasitic liver fluke, is the primary aetiological agent of clonorchiasis, a disease predominantly characterized by liver-related clinical manifestations. Currently, research on the complete mitochondrial (mt) genome of local C. sinensis populations remains inadequate. Thus, in this study, we sequenced and annotated the mt genome of fish-borne C. sinensis (Cs-c2) from Changchun, Jilin Province, China, a strain not previously described. This mt genome is 14,136 bp in length and harbours 12 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNAs (rRNAs), and a single control region (CR). We constructed a maximum likelihood (ML) phylogenetic tree using concatenated ND5, ND6, and ND1 from protein-coding genes (PCGs) of the C. sinensis mitochondrial genome (mt genome). This tree more clearly differentiated C. sinensis strains from three geographical regions (China, Russia, and South Korea) and distinguished Opisthorchiidae from two closely related families (Fasciolidae and Dicrocoeliidae). Additionally, we constructed an ML phylogenetic tree using concatenated ND4, ND5, ND1, ND2, and COX1 from the PCGs of digenean (Digenea) mt genomes. This approach—utilizing multiple high-resolution PCGs with evolutionary rates distinct from those of the mt genome—yielded robust clustering for multiple suborders and 13 families within Digenea and provided new molecular evidence for intergeneric relationships within the suborder Plagiorchiata of Digenea. These findings serve as important references for future research on the differentiation of closely related geographical strains of digeneans, as well as for studies on molecular taxonomy and population genetics. Full article

Background/Objectives: Pothos chinensis is commonly used as traditional medicine in China and India. Codon usage analysis is a good way to understand plants’ evolution. However, there is no report about the codon usage bias of chloroplast genomes in P. chinensis. Methods: In this study, the chloroplast genome of the medicinal plant P. chinensis was newly obtained. Comparative analyses, DNA barcoding investigation, codon usage bias, and phylogenetic reconstruction were conducted to reveal the chloroplast genome characteristics of P. chinensis. Results: The length of the chloroplast genome of P. chinensis was 165,165 bp. A total of 134 genes were annotated, i.e., 90 protein-coding genes, 36 transfer RNA genes, and eight ribosomal RNA genes. Compared to its sister group Anthurium andraeanum, the length of the large single-copy region (LSC) had been expanded, while the small single-copy region (SSC) had been contracted. Within P. chinensis and P. scandens there were no obvious differences in the length of LSC, SSC, and two inverted repeat regions. Based on Pi values, seven hypervariable regions of whole plastomes were identified. The analysis of codons showed that an average frequency of the 50 candidate genes was 35.30%, and these genes preferred A/U-ending codons. The average effective number of codon (ENC) value was 45.49, which indicated weak codon usage bias. ENCs had a highly significant positive correlation with GC3. Fourteen optimal codons had been identified, 11 of which ended with A/U. The results of the neutrality plot, ENC-plot, and PR2-plot analysis indicated that natural selection might have a significant impact on codon usage patterns. Conclusions: Taken together, our study unraveled the codon usage patterns in P. chinensis and provided valuable genetic information for the genus Pothos. Full article

Acute myeloid leukemia (AML) proliferation is significantly influenced by the interactions between leukemia blasts and the bone marrow (BM) microenvironment. Specifically, bone marrow mesenchymal stem cells (BMSCs) derived from AML patients (AML-MSCs) are known to support leukemia growth and facilitate disease progression. Studies have demonstrated that the transfer of mitochondria from MSCs to AML blasts not only aids in disease progression but also contributes to chemotherapy resistance. Furthermore, BM stromal cells can trigger a metabolic shift in malignant cells from mitochondrial respiration to glycolysis, which enhances both growth and chemo-resistance. This study focuses on identifying transcriptional and metabolic alterations in AML-MSCs to uncover potential targeted therapies for AML. We employed RNA sequencing and microarray analysis on MSCs cocultured with leukemic cells (MLL-AF9) and on MSCs isolated from both non-leukemic and MLL-AF9 leukemic mice. The Gene Set Enrichment Analysis (GSEA) indicated a significant downregulation of gene sets associated with oxidative phosphorylation and glycolysis in AML-MSCs. Furthermore, coculture of MSCs from wild-type mice (WT-MSCs) and a healthy donor individual (HD-MSCs) with AML cells demonstrated reduced oxidative phosphorylation and glycolysis. These metabolic changes were consistent in AML-MSCs derived from both leukemic mice and patients. Our results indicate that AML cells diminish the metabolic capacity of MSCs, specifically targeting oxidative phosphorylation and glycolysis. These findings suggest potential metabolic vulnerabilities that could be exploited to develop more effective therapeutic strategies for AML. Full article

Pyrola decorata Andres (P. decorata) is a traditional medicinal plant in China. However, its chloroplast genome and the deep evolutionary relationships among its genus remain unexplored. This study identified the samples as P. decorata using morphological observations from Flora of China (FOC) and ITS sequences. It is the first to analyze the complete chloroplast genome of P. decorata using Illumina and Nanopore sequencing technologies, confirming a typical chloroplast dumbbell structure. The chloroplast DNA (cpDNA) of P. decorata is 179,999 bp in length, consisting of a large single copy (LSC) (62.3% of total length (112,150 bp)), a small single copy (SSC) (6.5% of total length (11,701 bp)), and two inverted repeat regions (IRA and IRB) (31.2% combined (28,074 bp × 2)). Functional annotation revealed 128 genes: 77 conserved coding sequences (CDS) genes, 43 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Phylogenetic analysis placed P. decorata, Pyrola atropurpurea (P. atropurpurea), Pyrola rotundifolia (P. rotundifolia), and Chimaphila japonica within Group I, with P. decorata exhibiting the closest chloroplast genomic affinity to P. atropurpurea. These findings integrate morphological and molecular evidence to facilitate further identification, classification, and evolutionary analysis of this genus. Full article

Upon transferal from plastic to Matrigel, melanoma cells demonstrate growth in three dimensions and form de novo vascular networks—known as vasculogenic mimicry—that are characteristic of the stemness phenotype of aggressive tumors. It has been reported that during malignant transformation, stress, or differentiation, the long-range inter-chromosomal interactions between numerous developmental genes and nucleoli are changed. The aim of this work was to study the potential mechanisms behind the development of the vasculogenic mimicry phenotype in melanoma cells and whether the formation of these 3D structures is connected with the reorganization of inter-chromosomal contacts of rDNA clusters. Here, we show that after 15 h of growth on Matrigel, and following the formation of the vasculogenic mimicry phenotype, dramatic changes occur in Mel Z cells in rDNA contacts with different genomic regions that possess mainly developmental genes. Approximately 400 genes that retained stable contacts with nucleoli were co-expressed with different lincRNAs and were highly associated with H3K27me3 marks and simultaneously regulated by different transcription factors. These genes are involved in development and cell adhesion and may control the basic stage of differentiation. The genes that acquired or increased contacts with rDNA clusters during growth on Matrigel are associated with cell morphogenesis, cell junctions, and the cytoskeleton. Here, we present the first evidence that nucleoli may be involved in both the activation and repression of particular groups of developmental rDNA-contacting genes in melanoma cells forming the vasculogenic mimicry phenotype. We conclude that the inter-chromosomal interactions between developmental genes and rDNA clusters are dynamic, and that nucleoli play an important role in the development of vasculogenic mimicry and stemness phenotypes in aggressive tumor genes. Full article

Background: Aspidophorodon Verma, 1967 (Macrosiphini: Aphidinae), is a genus within Aphididae (aphids) with ecological importance and a unique distribution, but there is a lack of mitogenomic data on the evolutionary relationships within this genus, hindering a comprehensive understanding of its evolutionary history. Methods: In this study, we present the complete mitochondrial genome sequence and features of Aspidophorodon indicum (David, Rajasingh & Narayanan, 1972) (Hemiptera: Aphididae) and further infer its phylogenetic position based on the complete mitochondrial genome sequence. Results: The complete mitochondrial genome of A. indicum is 17,161 bp in length, including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, a control region, and a repeat region between trnE and trnF. Phylogenetic analyses based on complete mitochondrial genomes of Aphidinae indicated that the two constituent tribes, Macrosiphini and Aphidini, are monophyletic. Aspidophorodon was robustly clustered with the members of Pterocomma and Cavariella. Together, these three genera form the most basal clade within Macrosiphini. Conclusions: The complete mitogenome of A. indicum contains multiple conserved features relative to other aphids, including gene order, nucleotide composition, codon bias, and repeat region. The phylogenetic relationships within Macrosiphini reported here are consistent with previous studies. Our results provide new insights into the phylogenetic position of the genus Aspidophorodon. Full article

Oncolytic herpes simplex virus (oHSV) is a promising cancer immunotherapy that induces tumor cell lysis and stimulates anti-tumor immunity. Our previous single-cell RNA sequencing analysis of oHSV-treated medulloblastoma tumors revealed expansion and activation of tumor-infiltrating dendritic cells (DCs), and direct oHSV infection of DCs within the brain. While the therapeutic effects of oHSVs have been primarily attributed to tumor cell infection, we hypothesize that direct infection of DCs also contributes to therapeutic efficacy by promoting DC maturation and immune activation. Although the oHSV infection in DCs was abortive, it led to increased expression of major histocompatibility complex (MHC) class I/II and co-stimulatory molecules. oHSV-infected DCs activated naïve CD4⁺ and CD8⁺ T cells, inducing expression of CD69 and CD25. These primed T cells exhibited enhanced cytotoxicity against CT-2A glioma cells. Adoptive transfer of oHSV-infected DCs via subcutaneous injection near inguinal lymph nodes delayed tumor growth in a syngeneic CT-2A glioma model, independent of tumor viral replication and lysis. Mechanistically, our in vitro studies demonstrate that oHSV can directly infect and functionally activate DCs, enabling them to prime effective anti-tumor T cell responses. This study highlights the anti-tumor potential of leveraging oHSV-infected DCs to augment viroimmunotherapy as a cancer therapeutic. Full article

Hippophae rhamnoides ssp. sinensis Rousi a species of significant ecological and economic value that is native to the Qinghai–Tibet Plateau and arid/semi-arid regions. Investigating the mitochondrial genome can elucidate stress adaptation mechanisms, population genetic structure, and hybrid evolutionary history, offering molecular insights for ecological restoration and species conservation. However, the genetic information and evolutionary mechanisms of its mitochondrial genome remain poorly understood. This study aimed to assemble the complete mitochondrial genome of H. rhamnoides L. ssp. sinensis using Illumina sequencing, uncovering its structural features, evolutionary pressures, and environmental adaptability and addressing the research gap regarding mitochondrial genomes within the Hippophae genus. The study assembled a 454,444 bp circular mitochondrial genome of H. rhamnoides ssp. sinensis, with a GC content of 44.86%. A total of 73 genes and 3 pseudogenes were annotated, with the notable absence of the rps2 gene, which is present in related species. The genome exhibits significant codon usage bias, particularly with high-frequency use of the alanine codon GCU and the isoleucine codon AUU. Additionally, 449 repetitive sequences, potentially driving genome recombination, were identified. Our evolutionary pressure analysis revealed that most genes are under purifying selection, while genes such as atp4 and nad4 exhibit positive selection. A nucleotide diversity analysis revealed that the sdh4 gene exhibits the highest variation, whereas rrn5 is the most conserved. Meanwhile, phylogenetic analysis showed that H. rhamnoides ssp. sinensis from China is most closely related to Hippophae tibetana, with extensive homologous sequences (49.72% of the chloroplast genome) being identified between the chloroplast and mitochondrial genomes, indicating active inter-organellar gene transfer. Furthermore, 539 RNA editing sites, primarily involving hydrophilic-to-hydrophobic amino acid conversions, were predicted, potentially regulating mitochondrial protein function. Our findings establish a foundation for genetic improvement and research on adaptive evolutionary mechanisms in the Hippophae genus, offering a novel case study for plant mitochondrial genome evolution theory. Full article

Microbial communities, as critical functional components of riverine ecosystems, play a pivotal role in biogeochemical cycles and water quality regulation. The South-to-North Water Diversion Middle Route Project (SNWD-MRP) is a major cross-basin water transfer initiative, and bacteria are essential for the stability of water quality in the project. This study employed environmental DNA (eDNA) metabarcoding targeting the 16S rRNA gene to investigate spatiotemporal variations in water quality and bacterial communities along the SNWD-MRP during summer and winter. Integrated analyses, including redundancy analysis (RDA), Mantel tests, and ecological network modeling, were applied to unravel the driving mechanisms of microbial succession. The water quality along the SNWD-MRP is generally classified as Grade I, with significant seasonal variations in water quality parameters and microbial community composition. In the summer, higher temperatures lead to an increased abundance of cyanobacteria. In contrast, during the winter, lower water temperatures and higher dissolved oxygen levels result in the dominance of Pseudomonas and Bacillota species. RDA identified the permanganate index as the primary driver of microbial composition across seasons, with total phosphorus and total nitrogen having a greater influence in winter. Mantel tests highlighted significant correlations between Cyanobacteria and total phosphorus during winter. Ecological network analysis revealed that the complexity and connectivity of the winter network increased, likely due to suitable nutrient levels rendering the microbial network more complex and stable. These findings underscore the synergistic effects of temperature and nutrient availability on microbial succession, providing actionable insights for optimizing water quality management and ecological stability in large-scale water diversion systems. Full article

Background: Centella asiatica, a medicinally important species that is rich in bioactive compounds, lacks a characterized mitochondrial genome, despite nuclear and chloroplast assemblies. We sequenced and annotated its mitochondrial genome to elucidate its genetic foundations and evolutionary mechanisms. Methods: Assembly using Illumina short-reads and Nanopore long-reads was used to characterize the mitochondrial genome. Analyses included structural characterization, codon usage bias, repetitive sequences, horizontal gene transfer (HGT), collinearity, and phylogeny. The resulting tissue-specific (root, stem, and leaf) long non-coding RNA (lncRNA) profiles identified RNA editing sites. Results: The complete mitochondrial genome (249,777 bp, 45.5% GC) comprises three circular contigs encoding 51 genes (33 protein-coding, 15 tRNA, and 3 rRNA). Comparative genomics revealed synteny with the Apiaceae family of plants and evidence of HGT. Phylogenetic analysis resolved taxonomic relationships within Apiales. We predicted that 547 RNA editing sites would be identified in its protein-coding genes. Tissue profiling identified 725 (root), 711 (stem), and 668 (leaf) editing sites, with >71% concordance to predictions. RNA editing-generated cryptic promoters/terminators occur in mitochondrial core function genes (e.g., ATP synthase, cytochrome c reductase/oxidase, ribosome large subunit, and cytochrome c biogenesis), exhibiting a lower frequency in the leaves compared to the roots and stems. Conclusions: We provide the first complete mitochondrial genome assembly for C. asiatica, delineating its complex structure, tissue-modulated RNA editing, and evolutionary trajectory. This high-quality genomic resource establishes a foundation for molecular evolutionary studies and enhances the genomic toolkit for this pharmacologically significant species. Full article