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Background Mitochondrial DNA sequences are used for inter- and intra-specific comparison analysis in ecological studies. Instead of using short regions as marker sequences, analyzing longer regions, such as whole mitochondrial DNA sequences, can improve the accuracy of such studies by increasing the likelihood of detecting species or specific sequences. However, current methods for sequencing whole mitochondrial DNA require primer design for each target species or long fragments of genomic DNA as a PCR template. We developed a method and accompanying tool for PCR-based long-read sequencing of whole mitochondrial DNA, named MitoCOMON, which is applicable to wide-target taxonomic clades and partially digested template DNA. Results PCR amplification of whole mitochondrial DNA as four fragments facilitates the successful assembly of the whole mitochondrial DNA sequence, even when a sample is a mixture of multiple species or partially degraded. The tool that we developed consists of two modules that can design a primer set for species in a target taxonomic clade and assemble the whole mitochondrial DNA sequence from amplicons which were amplified using the designed primer set. Primer sets were designed for mammal and bird species, which showed a high success rate for whole mitochondrial DNA sequencing with high sequence accuracy. Multiple whole mitochondrial DNA sequences were also assembled from samples mixed with the genomic DNA of several species without forming chimeric sequences. In addition to the accuracy, some assembled sequences also retained a long duplication at the D-loop region, suggesting that the method addresses large rearrangements. Compared with a method that amplifies the whole mitochondrial DNA as a single amplicon, our method was effective for partially degraded samples. Conclusions Our method and accompanying tool, named MitoCOMON, enables an easier acquisition of whole mitochondrial DNA sequences from samples with some DNA degradation without designing species-specific primers. This approach can enhance the accessibility of mitochondrial genomic data and is expected to improve the resolution of ecological analyses, including accurate species identification and individual-level discrimination.

Two veterinary personnel in Japan were infected with severe fever with thrombocytopenia syndrome virus (SFTSV) while handling a sick cat. Whole-genome sequences of SFTSV isolated from the personnel and the cat were 100% identical. These results identified a nosocomial outbreak of SFTSV infection in an animal hospital without a tick as a vector.

Environmental DNA (eDNA) enables non‐invasive assessment of aquatic biodiversity. Because eDNA is typically diluted and fragmented, most applications rely on short mitochondrial amplicons, which often fail to resolve closely related species and do not support individual‐level inference. However, intact or near‐full‐length mitochondrial genomes may persist in some waters. Advances in long‐read sequencing (LRS) may enable recovery of whole mitogenomes from eDNA; however, current protocols are not optimised to detect or utilise longer fragments. To address this, we developed an LRS workflow to reconstruct complete mitogenomes from eDNA. Using aquarium water from Carassius auratus and Oryzias latipes, we optimised DNA extraction and filter pore sizes to maximise recovery of high‐molecular‐weight target DNA. We then evaluated two strategies: PCR‐free sequencing of native eDNA and long‐range PCR followed by nanopore sequencing. Finally, using single‐fish aquaria, we compared the resulting full‐length sequences across samples to assess the feasibility of non‐invasive, mitogenome‐wide polymorphism analysis. Using the selected extraction method and filters (1.2‐μm pore size), PCR‐free datasets from single‐fish C. auratus aquaria yielded hundreds of mitochondrial reads, including >16 kb molecules, enabling de novo assembly of full‐length mitogenomes in several cases. PCR‐based datasets sometimes produced >100,000 mitochondrial reads, and, after careful preprocessing, these data could also be assembled successfully. Assemblies closely matched tissue‐derived references, and cross‐sample comparisons resolved two C. auratus haplotypes. These results reveal that full‐length mitogenomes can be reconstructed from eDNA using both PCR‐free and PCR‐based strategies under appropriate conditions. Our results show that LRS can recover near‐complete mitogenomes directly from eDNA, including through PCR‐free workflows. This capability advances eDNA‐based genetic polymorphism analysis, supporting aquaculture management and non‐invasive genetic monitoring of captive conservation populations. With further refinement and field validation, it could also enable detailed assessment of genetic diversity in natural populations.

Plectranthus hadiensis (Lamiaceae) is recognized for its rich terpene content and potential applications in agriculture, medicine, and aromatherapy. Terpenes are major constituents of P. hadiensis essential oil, yet its terpene synthase (TPS) genes remain insufficiently characterized. In this study, we assembled a de novo transcriptome from RNA-seq data generated from leaf, stem, and root tissues and identified 26 TPS genes. Phylogenetic analysis classifies these genes into five TPS subfamilies (TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g), broadly associated with sesquiterpene, monoterpene, and diterpene biosynthesis. Expression profiling revealed apparent tissue specificity; notably, PhTPS1 showed high transcript abundance in the leaf and stem. BLASTP analysis indicated that PhTPS1 is closely related to Lamiaceae monoterpene synthases, with the top hit being a rosemary ( Salvia rosmarinus ) limonene synthase. Heterologous expression of PhTPS1 in yeast, followed by headspace gas chromatography-mass spectrometry, detected limonene, confirming PhTPS1 as a functional limonene synthase. To our knowledge, this is the first limonene synthase gene functionally characterized in P. hadiensis . Collectively, these findings provide a curated TPS genes catalog with their tissue-specific expression patterns and identify PhTPS1 as a promising target for breeding and metabolic engineering to enhance limonene yield.

Neuroendocrine carcinoma (NEC) is a highly aggressive subtype of the neuroendocrine tumor with an extremely poor prognosis. We have previously conducted a comprehensive genomic analysis of over 100 cases of NEC of the gastrointestinal system (GIS‐NEC) and unraveled its unique and organ‐specific genomic drivers. However, the epigenomic features of GIS‐NEC remain unexplored. In this study, we have described the epigenomic landscape of GIS‐NEC and small cell lung carcinoma (SCLC) by integrating motif enrichment analysis from the assay of transposase‐accessible chromatin sequencing (ATAC‐seq) and enhancer profiling from a novel cleavage under targets and tagmentation (CUT&Tag) assay for H3K27ac and identified ELF3 as one of the super‐enhancer–related transcriptional factors in NEC. By combining CUT&Tag and knockdown RNA sequencing for ELF3, we uncovered the transcriptional network regulated by ELF3 and defined its distinctive gene signature, including AURKA, CDC25B, CLDN4, ITGB6, and YWAHB. Furthermore, a loss‐of‐function assay revealed that ELF3 depletion led to poor cell viability. Finally, using gene expression of clinical samples, we successfully divided GIS‐NEC patients into two subgroups according to the ELF3 signature and demonstrated that tumor‐promoting pathways were activated in the ELF3 signature–high group. Our findings highlight the transcriptional regulation of ELF3 as an oncogenic transcription factor and its tumor‐promoting properties in NEC. Multiorgan comprehensive epigenomic analysis identified ELF3 as a super‐enhancer–associated transcription factor and revealed its oncogenic properties in neuroendocrine carcinoma.

Integration of human papillomavirus (HPV) into the host genome drives HPV-positive head and neck squamous cell carcinoma (HPV + HNSCC). Whole-genome sequencing of 51 tumors revealed intratumor heterogeneity of HPV integration, with 44% of breakpoints subclonal, and a biased distribution of integration breakpoints across the HPV genome. Four HPV physical states were identified, with at least 49% of tumors progressing without integration. HPV integration was associated with APOBEC-induced broad genomic instability and focal genomic instability, including structural variants at integration sites. HPV + HNSCCs exhibited almost no smoking-induced mutational signatures. Heterozygous loss of ataxia-telangiectasia mutated ( ATM ) was observed in 67% of tumors, with its downregulation confirmed by single-cell RNA sequencing and immunohistochemistry, suggesting ATM haploinsufficiency contributes to carcinogenesis. PI3K activation was the major oncogenic mutation, with JAK-STAT activation in tumors with clonal integration and NF-kappa B activation in those without. These findings provide valuable insights into HPV integration in HPV + HNSCC. The integration of human papillomavirus (HPV) drives HPV-positive head and neck squamous cell carcinoma (HPV +  HNSCC). Here, the authors perform genomic analysis to investigate HPV integration in patients with HPV +  HNSCC, identifying intratumor heterogeneity and biased distribution of HPV integration.

Verrucous carcinoma (VC) is a rare variant of squamous cell carcinoma. Although VC is considered radioresistant, concrete evidence for this is absent. We obtained data on VC treated with surgery or radiation from the Surveillance, Epidemiology, and End Results database. Treatment selection bias was reduced by propensity score matching. Overall survival (OS) and disease-specific survival (DSS) rates were estimated using the Kaplan-Meier method. Hazard ratios (HRs) were estimated using Cox proportional hazards models. Five-year OS rates in the radiation and surgery groups were 72.7% and 72.0%, respectively (P = 0.111); five-year DSS rates in the same were 86.7% and 88.4%, respectively (P = 0.234). HRs of radiation compared with surgery were 1.68 (95% confidence interval (CI), 0.96-2.95) for OS and 1.95 (95% CI, 0.69-5.53) for DSS. Similar prognoses were observed in patients with VC treated with radiation and surgery. VC can be treated using radiation.

Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor with dismal prognosis. Recently, molecular subtypes of SCLC have been defined by the expression status of ASCL1, NEUROD1, YAP1, and POU2F3 transcription regulators. ASCL1 is essential for neuroendocrine differentiation and is expressed in the majority of SCLC. Although previous studies investigated ASCL1 target genes in SCLC cells, ASCL1‐mediated regulation of miRNAs and its relationship to molecular subtypes remain poorly explored. Here, we performed genome‐wide profiling of chromatin modifications (H3K27me3, H3K4me3, and H3K27ac) by CUT&Tag assay and ASCL1 knockdown followed by RNA sequencing and miRNA array analyses in SCLC cells. ASCL1 could preferentially regulate genes associated with super‐enhancers (SEs) defined by enrichment of H3K27ac marking. Moreover, ASCL1 positively regulated several SE‐associated miRNAs, such as miR‐7, miR‐375, miR‐200b‐3p, and miR‐429, leading to repression of their targets, whereas ASCL1 suppressed miR‐455‐3p, an abundant miRNA in other molecular subtypes. We further elucidated unique patterns of SE‐associated miRNAs in different SCLC molecular subtypes, highlighting subtype‐specific miRNA networks with functional relevance. Notably, we found apparent de‐repression of common target genes of different miRNAs following ASCL1 knockdown, suggesting combinatorial action of multiple miRNAs underlying molecular heterogeneity of SCLC (e.g., co‐targeting of YAP1 by miR‐9 and miR‐375). Our comprehensive analyses provide novel insights into SCLC pathogenesis and a clue to understanding subtype‐dependent phenotypic differences. ASCL1 preferentially regulates genes and miRNAs associated with super‐enhancers (SEs). SCLC molecular subtypes show unique patterns of SE‐associated miRNAs. ASCL1‐induced SE‐associated miRNAs cooperatively regulate target genes; for example, miR‐9 and miR‐375 co‐target YAP1.

Circulating tumor DNA (ctDNA), which circulates in the blood after being shed from cancer cells in the body, has recently gained attention as an excellent tumor marker. The purpose of this study was to evaluate whether ct human papillomavirus (HPV) 16 DNA (ctHPV16DNA) levels were associated with quantitative PET parameters in patients with HPV-positive head and neck (HN) squamous cell carcinoma (SCC). Fifty patients with oropharyngeal SCC (OPSCC) and 5 with SCC of unknown primary (SCCUP) before treatment were included. They all underwent blood sampling to test ctHPV16DNA levels and FDG PET-CT examinations. Quantitative PET parameters included SUVmax, metabolic tumor volume (MTV), MTV of whole-body lesions (wbMTV), and 56 texture features. ctHPV16DNA levels were compared to texture features of primary tumors in OPSCC patients (Group A) or the largest primary or metastatic lymph node lesions in OPSCC and SCCUP patients (Group B) and to other PET parameters. Spearman rank correlation test and multiple regression analysis were used to confirm the associations between ctHPV16DNA levels and PET parameters. ctHPV16DNA levels moderately correlated with wbMTV, but not with SUVmax or MTV in Groups A and B. ctHPV16DNA levels exhibited a weak negative correlation with low gray-level zone emphasis in Groups A and B. Multiple regression analysis revealed that wbMTV and high gray-level zone emphasis were the significant factors for ctHPV16DNA levels in Group B. These results were not observed in Group A. This study demonstrated that ctHPV16DNA levels correlated with the whole-body tumor burden and tumor heterogeneity visualized on FDG PET-CT in patients with HPV-positive HNSCC.

Plant hormones have been identified to be versatile signaling molecules essential for plant growth, development, and stress response. Their content levels vary depending on the species, and they also change in response to any external stimuli. Thus, simultaneous quantification of multiple plant hormones is required to understand plant physiology. Sensitive and quantitative analysis using liquid chromatography-linked mass spectrometry (LC-MS/MS) has been used in detecting plant hormones; however, quantification without stable isotopes is yet to be established. In this study, we quantified seven representative plant hormones of Lotus japonicus , which is a model legume for standard addition method. Accurate masses for monoisotopic ions of seven phytohormones were determined for high-resolution mass spectrometry (HR-MS). Selected ion monitoring (SIM) mode based on accurate masses was used in detecting phytohormones in the roots, stems, and leaves. Evaluation of matrix effects showed ion suppression ranging from 10.2% to 87.3%. Both stable isotope dilution and standard addition methods were able to detect plant hormones in the roots, stems, and leaves, with no significant differences in using both approaches and thus a standard addition method can be used to quantify phytohormones in L . japonicus . The method will be effective, especially when stable isotopes are not available to correct for matrix effects.