Variation In Copy Number Research Articles

Generation of a novel constitutive smooth muscle cell-specific Myh11 -driven Cre mouse model.

Dysfunction in either embryonic or postnatal vascular smooth muscle cells (SMCs) significantly contributes to the progression of various cardiovascular diseases. Therefore, elucidating the molecular mechanisms governing VSMC development and homeostasis is crucial. MYH11 is the most reliable lineage gene for SMCs and has been utilized to develop tamoxifen-inducible Cre driver lines for achieving SMC-specific gene manipulation by crossing with mice carrying the lox P -flanked gene, particularly in adult mice. For studies involving SMCs during embryogenesis, the commonly used constitutive Cre driver is controlled by the Tagln ( Sm22α ) promoter. However, this Cre driver exhibits activity in multiple non-SMC populations, including cardiomyocytes and skeletal muscle precursors, introducing confounding effects. Additionally, most existing SMC-specific Cre drivers are generated using a transgenic approach, raising concerns about random site integration and variable gene copy numbers. To address these limitations, we report a novel Cre mouse model generated by knock-in (KI) of a nuclear-localized Cre recombinase into the Myh11 gene locus using homologous recombination. We confirmed that the Cre activity precisely recapitulates endogenous Myh11 expression by crossing with Rosa26 mTmG or tdTomato reporter mice. Moreover, Myh11 -driven Cre can efficiently delete the floxed allele of the transcription factor Tead1 specifically in SMCs. The Tead1 SMC-specific knockout mice did not exhibit an overt phenotype, thereby circumventing the embryonic lethal phenotype mediated by Tagln -driven Cre, as we previously reported. These findings establish this novel Cre driver line as a robust tool for tracing the Myh11 -positive SMC lineage and manipulating gene function specifically in SMCs during embryonic development in mice.

The heterogeneity of breast cancer metastasis: a bioinformatics analysis utilizing single-cell RNA sequencing data.

Breast cancer is a common malignancy in women, and its metastasis is a leading cause of cancer-related deaths. Single-cell RNA sequencing (scRNA-seq) can distinguish the molecular characteristics of metastasis and identify predictor genes for patient prognosis. This article explores gene expression in primary breast cancer tumor tissue against metastatic cells in the lymph node and liver using scRNA-seq. Breast cancer scRNA-seq data from the Gene Expression Omnibus were used. The data were processed using R and the Seurat package. The cells were clustered and identified using Metascape. InferCNV is used to analyze the variation in copy number. Differential expression analysis was conducted for the cancer cells using Seurat and was enriched using Metascape. We identified 18 distinct cell clusters, 6 of which were epithelial. CNV analysis identified significant alterations with duplication of chromosomes 1, 8, and 19. Differential gene analysis resulted in 17 upregulated and 171 downregulated genes for the primary tumor in the primary tumor vs. liver metastasis comparison and 43 upregulated and 4 downregulated genes in the primary tumor in the primary tumor vs lymph node metastasis comparison. Several enriched terms include Ribosome biogenesis, NTP synthesis, Epithelial dedifferentiation, Autophagy, and genes associated with epithelial-to-mesenchymal transitions. No single gene or pathway can clearly explain the mechanisms behind tumor metastasis. Several mechanisms contribute to lymph node and liver metastasis, such as the loss of differentiation, epithelial-to-mesenchymal transition, and autophagy. These findings necessitate further study of metastatic tissue for effective drug development.

INVESTIGATION OF THE EVOLUTION OF SABATH GENE FAMILY IN CASSAVA <i>(Manihot esculenta)</i> REVEALS ITS POTENTIAL ROLE IN GROWTH AND DEVELOPMENT

SABATH is one class of enzyme belonging to the class of methyltransferase, playing a crucial role in plant defense and stress response mechanisms. Despite its importance, no systematic analysis of the expansion of the SABATH gene family in cassava (Manihot esculenta) has been reported up to date. In this study, we investigated the SABATH gene family in cassava, revealing their random distribution across 18 chromosomes, with variable numbers of gene copies per chromosome. According to gene duplication analysis, five duplication events were identified, primarily segmental duplications, indicating their evolutionary significance. The Ka/Ks ratio analysis indicated that most duplicated genes are under negative selection, preserving their functions, while one pair showed signs of positive selection, suggesting adaptive benefits. Gene structure analysis showed diverse exon counts, primarily three or four. Expression profiling across 11 cassava tissues demonstrated tissue-specific expression patterns, with some genes highly or exclusively expressed in specific tissues such as root apical meristems, embryogenic calli, and fibrous roots, implying distinct functional roles in cassava growth and development. Overall, this study provides valuable insights into the evolution and functional diversity of the SABATH gene family in cassava and identifies candidate genes for further functional characterization.

A Homoplasmic MT-TV Mutation Associated with Mitochondrial Inheritance of Hereditary Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) is characterized by progressive lower limb weakness and spasticity, with unknown genetic cause in many cases. To identify novel genetic causes of HSP. Phenotypic characterization, genetic screening, transcriptome sequencing, and peroneal nerve biopsy were conducted in a Chinese HSP family. We found a homoplasmic MT-TV (mitochondrial tRNAVal) mutation, m.1661A > G, present in all affected individuals across four generations of a family with complex HSP. Fourth-generation affected individuals displayed earlier onset, likely due to presumptive anticipation, and greater symptom severity, potentially caused by decreased mitochondrial DNA (mtDNA) copy number. Upregulation of mitochondrial autophagy genes in these patients suggested that MT-TV mutations could lead to reduced mtDNA copy number. Neural biopsies revealed ultrastructural abnormalities in myelin and mitochondria. The rare MT-TV m.1661A > G mutation is associated with HSP. Variations in mtDNA copy number may play a causal role in differences among clinical phenotypes. © 2024 International Parkinson and Movement Disorder Society.

Genotyping sequence-resolved copy-number variations using pangenomes reveals paralog-specific global diversity and expression divergence of duplicated genes.

Copy-number variable (CNV) genes are important in evolution and disease, yet sequence variation in CNV genes is a blindspot for large-scale studies. We present a method, ctyper, that leverages pangenomes to produce copy-number maps with allele-specific sequences containing locally phased variants of CNV genes from NGS reads. We extensively characterized accuracy and efficiency on a database of 3,351 CNV genes including HLA , SMN , and CYP2D6 as well as 212 non-CNV medically-relevant challenging genes. The genotypes capture 96.5% of underlying variants in new genomes, requiring 0.9 seconds per gene. Expression analysis of ctyper genotypes explains more variance than known eQTL variants. Comparing allele-specific expression quantified divergent expression on 7.94% of paralogs and tissue-specific biases on 4.7% of paralogs. We found reduced expression of SMN-1 converted from SMN-2, which potentially affects diagnosis of spinal muscular atrophy, and increased expression of a duplicative translocation of AMY2B . Overall, ctyper enables biobank-scale genotyping of CNV and challenging genes.

Centromere Landscapes Resolved from Hundreds of Human Genomes.

High-fidelity (HiFi) sequencing has facilitated the assembly and analysis of the most repetitive region of the genome, the centromere. Nevertheless, our current understanding of human centromeres is based on a relatively small number of telomere-to-telomere assemblies, which has not yet captured its full diversity. In this study, we investigated the genomic diversity of human centromere higher order repeats (HORs) via both HiFi reads and haplotype-resolved assemblies from hundreds of samples drawn from ongoing pangenome-sequencing projects and reprocessed them via a novel HOR annotation pipeline, HiCAT-human. We used this wealth of data to provide a global survey of the centromeric HOR landscape; in particular, we found that 23 HORs presented significant copy number variability between populations. We detected three centromere genotypes with unbalanced population frequencies on chromosomes 5, 8, and 17. An inter-assembly comparison of HOR loci further revealed that while HOR array structures are diverse, they nevertheless tend to form a number of specific landscapes, each exhibiting different levels of HOR subunit expansion and possibly reflecting a cyclical evolutionary transition from homogeneous to nested structures and back.

Comparative Efficacy of Current and Emerging Therapies for Cutaneous and Visceral Leishmaniasis: A Global Perspective

Leishmaniasis treatment has faced challenges with pentavalent antimonials, historically used but now showing resistance and high failure rates. The World Health Organization (WHO) recommended higher doses, but resistance in Nepal and misuse persist. Antimonials are no longer the preferred treatment for visceral leishmaniasis (VL) in the Indian subcontinent but are still used for cutaneous leishmaniasis (CL). Amphotericin B, especially liposomal amphotericin B (L-AmB), has become the preferred treatment for VL in the Indian subcontinent, with high efficacy and shorter treatment courses. Genetic insights into leishmaniasis reveal that parasite species and host immune responses are key factors in disease variation. Genetic analysis of atypical parasite isolates shows that genetic variations can lead to different disease patterns. Whole genome sequencing and proteomic analyses offer fresh insights into how parasite genes and proteins impact drug resistance, post-kala-azar dermal leishmaniasis (PKDL), and unusual disease presentations. Variations in hereditary unit copy numbers and single nucleotide polymorphisms contribute to genetic diversity and distinct tissue distribution, leading to distinct disease patterns. Genomic markers for drug resistance in Leishmania parasites include ATP-binding cassette (ABC) transporters, amino acid permease 3 (AAP3), and proteins like phosphoglycerate kinase (PGK) and mitogen-activated protein kinase (MAPK). These markers are associated with efflux of thiols and metals, antimony resistance, and protection against oxidative stress. Additionally, key proteins and enzymes in antioxidant defense mechanisms, such as iron superoxide dismutase-A (FeSOD-A), folate transporter 1 (FT1), and heat shock protein 83 (HSP83), play roles in drug resistance and parasite survival.

7397 Characterization of Sperm-Carried IGF2 Transcript Variants

Abstract Disclosure: R. Cannarella: None. M. Suryavanshi: None. A. Miller: None. S.D. Lundy: None. A.E. Calogero: None. Background: We have previously identified the expression of the highly-conserved paternally-imprinted insulin-like growth factor 2 (IGF2) gene in human spermatozoa and suggested its role in early embryo development. Although there are 5 different variants of the IGF2, there is no evidence of the variants expressed in human spermatozoa. Objective: The present basic research study was undertaken to characterize the IGF2 transcript variant(s) expressed in human spermatozoa and to develop a sensitive method that can distinguish variants based on quantitative polymerase chain reaction (qPCR). Methods: Two post-gradient sperm samples from healthy, fertile men with proven paternity were used. Granulosa cells (GCs) obtained after the oocyte retrieval procedure and subsequently isolated and purified from follicular fluid were used as controls. Each sample underwent RNA extraction, cDNA synthesis, and PCR. The PCR product was ligated with the pCR4-TOPO vector and used to transform TOPO10 one-shot electro-competent cells of E. coli DH-10α. For each sample, 32 white colonies (true positive clones selected by blue-white screening method) were grown per sample (total in 96). All 96 clones were fully characterized by Sanger sequencing and single nucleotide polymorphism (SNP) were detected. Variations in products from single IGF2 gene primers pair were recorded on Sybr green-based qPCR, such as variations in melting temperatures (Tm), product size, and number of copies. Results: We successfully isolated and identified 96 true positive colonies, which were confirmed as IGF2 cDNA clones. This comprehensive analysis revealed 11 distinct genetic variants within the cDNA clones. Specifically, these variants comprised 6 SNPs and 5 deletions, highlighting the diversity in the sequences. These findings were particularly significant in relation to the IGF2 primer employed in the study, as they indicated the presence of 11 different sequence variants. By Sybr green qPCR, clones derived from GCs and sperm samples predominantly showed an IGF2 transcript variant with Tms of 89.01°C, 88.86°C, and 88.71°C. In a surprising observation, the sperm samples exhibited three sperm-specific variants with a range of a Tm ranging from 89.01°C to 88.26°C. Furthermore, the deletions were characterized by a significantly lower Tm, ranging between 78.86°C and 71.11°C. Conclusion: The IGF2 transcript is intrinsically present in sperm samples, albeit with notable variations. The detected variants likely arise from SNPs within the transcripts and the deletions could be attributed to splicing activity during maturation or post-transcriptional modifications. To our knowledge, this is the first study targeting the IGF2 transcript variants in spermatozoa from fertile men. Basic research is needed to understand the implications of different sperm-specific IGF2 transcript variants on early embryo development. Presentation: 6/2/2024

Genome architecture of the heavy metal tolerant and accumulator Hirschfeldia incana: Insights from genome sequencing, assembly, and comparative analysis

Hirschfeldia incana L., a member of the Brassicaceae family commonly found in Mediterranean regions, is known for its capacity to withstand and accumulate heavy metals, particularly lead (Pb) both in soil environments and hydroponic systems. This plant has been used as a model to study plant responses to heavy metals. Nonetheless, the molecular mechanisms underlying its tolerance and heavy metal accumulation are not fully understood, partly because of the limited knowledge about its genome. In this study, the genome of H. incana was sequenced, assembled, characterized, and annotated. Approximately 8.6 Gpb of data were generated using Oxford Nanopore Technology (ONT), resulting in a genome assembly of 390 Mb, comprising 5196 contigs with an N50 exceeding 131 kb. The genome had a BUSCO score of 97.2 %, with 38,454 genes and a repetition content of 38.25 %. Subsequently, the assembled genome was annotated using several databases including GO, InterPro, MetaCyc, PANTHER, Pfam, Reactome, SUPERFAMILY, and KEGG. This annotation yielded 22,661 GO terms and 143 KEGG maps. A comparative genomic analysis between H. incana and six Brassicaceae species (five hyperaccumulators of heavy metals and one non-hyperaccumulator) was also conducted. This analysis revealed that H. incana shares a substantial proportion of orthologous genes (89.7 % of orthogroups) with six Brassicaceae species. The generated phylogenetic tree suggests that H. incana is closely related to B. juncea, B. napus, and B. oleracea, indicating a common ancestry and potentially shared genetic factors contributing to hyperaccumulation in these species. Moreover, the copy number of twenty-nine genes involved in heavy metal tolerance and accumulation mechanisms in H. incana and six brassicaceae were assayed. This analysis revealed that H. incana and other hyperaccumulator species possess a higher copy number of genes related to heavy metal tolerance than the sensitive plant A. thaliana. Notably, the variation in gene copy numbers highlights their potential role in the adaptation of H. incana to heavy metal stress. This study provides a comprehensive genomic framework that enhance our understanding of H. incana adaptation to heavy metal stress, and offers valuable data for further genomic investigations of the molecular mechanisms of heavy metal tolerance and accumulation in plants.

SNRPB2 in the pan-cancer landscape: A bioinformatics exploration and validation in hepatocellular carcinoma

Aberrant splicing is a significant contributor to gene expression abnormalities in cancer. SNRPB2, a component of U2 small nuclear ribonucleoprotein particles (snRNPs), contributes to the assembly of the spliceosome, the molecular machinery responsible for splicing. To date, few studies have investigated the role of SNRPB2 in tumorigenesis. We examined data sourced from various public databases, such as The Cancer Genome Atlas(TCGA), the Clinical Proteomic Tumor Analysis Consortium(CPTAC), and Gene Expression Omnibus(GEO). Our investigation included gene expression, genomic and epigenomic scrutiny, gene set enrichment assessment(GSEA), and immune cell infiltration evaluation. Furthermore, we performed empirical validation to ascertain the impact of SNRPB2 suppression on the proliferation and migration of liver cancer cells. Analysis of gene expression revealed widespread upregulation of SNRPB2 across a spectrum of cancer types, with heightened levels of SNRPB2 expression in numerous tumors linked to unfavorable prognosis. Genomic and epigenomic assessments revealed connections between SNRPB2 expression and variations in SNRPB2 copy number, DNA methylation patterns, and RNA modifications. Through gene set enrichment analysis, the involvement of SNRPB2 in vital biological processes and pathways related to cancer was identified. Furthermore, scrutiny of immune cell infiltration suggested a potential relationship between SNRPB2 and the tumor microenvironment, which was reinforced by multiple single-cell sequencing profiles. Subsequent experimental validation revealed that silencing SNRPB2 effectively impeded the proliferation and migration of liver cancer cells. Taken together, these findings underscore the prospective utility of SNRPB2 as a prognostic biomarker and a promising candidate for immunotherapy in cancer. It is necessary to engage in additional exploration into its underlying mechanisms and clinical treatment potential.

Comprehensive analysis of bulk, single-cell RNA sequencing, and spatial transcriptomics revealed IER3 for predicting malignant progression and immunotherapy efficacy in glioma

BackgroundAs part of stress-triggered molecules, immediate early response 3 (IER3) dysregulation has been reported to sustain pro-oncogenic pathways and precede malignant transformation. However, the role of IER3 in glioma pathology is ill-defined.MethodsImmunohistochemistry (IHC) assay and publicly available glioma datasets were used to calculate the IER3 expression level in glioma. Wound healing, invasion and cell counting kit-8 (CCK8) assays were applied to measure the cell viability and capacities of migration and invasion of glioma cells in vitro. The immunofluorescence (IF) assay was used to assess the expression associations of IER3 with CCL2 and TGFBI. Cox regression analysis and Kaplan-Meier (K-M) curve were introduced to compute the prognosis-predicting value of IER3. Variations in copy number (CNVs), single nucleotide (SNVs), and methylation profiles were analyzed to illustrate the epigenetic modifications of IER3. Gliomas were divided into two subgroups using the restricted cubic spline (RCS) method.Results IER3was overexpressed and hypomethylated in gliomas and significantly associated with the dismal prognosis of glioma samples. Samples in the high IER3 subgroup were characterized by increased infiltration of tumor-associated monocytes/macrophages (TAMMs), as well as the elevated sensitivity to Dabrafenib, an inhibitor of BRAF. In addition, this subgroup demonstrated a low mutation rate of IDH, high gain rates of BRAF, ELTD1, and PDGFA. Gliomas with relatively low IER3 expression demonstrated a less invasive subtype and were featured by favorable prognosis, increased response to immunotherapy, and adjuvant chemotherapy plus radiotherapy. The IF assay revealed that IER3 was co-localized and co-expressed with TGFBI. The glioma cells with small interfering RNA (siRNA)-silenced IER3 displayed lower migration, invasion, proliferation, and cell viability than the control group.ConclusionsIn this study, we identified IER3 upregulation as an essential biomarker that could assist in adjuvant therapy and prognosis prediction for gliomas.

Successful generation of fully human, second generation, anti-CD19 CAR T cells for clinical use in patients with diverse autoimmune disorders

BackgroundB-cell targeting chimeric antigen receptor (CAR) T-cell therapies, which lead to profound B-cell depletion, have been well-established in hematology-oncology. This deep B-cell depletion mechanism has prompted the exploration of their use in B-cell driven autoimmune diseases. We herein report on the manufacturing of KYV-101, a fully human anti-CD19 CAR T-cell therapy, derived from patients who were treated across a spectrum of autoimmune diseases. MethodsKYV-101 was manufactured from peripheral blood-derived mononuclear cells of 20 patients across 7 autoimmune disease types (neurological autoimmune diseases, n=13; rheumatological diseases, n=7). Patients ranged from 18–75 years of age. Duration of disease ranged from <1–23 years since diagnosis. Patients were heavily pretreated, and most were refractory to prior immunosuppressive treatments. Apheresis was collected across 9 sites, cryopreserved, and shipped to the manufacturing facility. Healthy donor apheresis samples were collected for manufacturing comparison. Manufacturing was performed using the CliniMACS Prodigy system. Cells were enriched for CD4+/CD8+ T cells, transduced with a 3rd generation lentiviral vector encoding the CAR, expanded in vitro, and harvested. Percent cell viability, T-cell purity, cellular expansion, and transduction efficiency were assessed. Activity was assessed using cytokine release assays for KYV-101 CAR T cells co-cultured with different CD19+/– target cell lines. ResultsKYV-101 was successfully manufactured for 100% of patients. Transduced cell populations were highly viable, with expansion ranging from 11–66 fold at Day 8, and were comparable across disease types. Healthy donor-derived controls displayed similar expansion ranges. High CAR expression and transduction rates were observed, ranging between 37–84% with low variation in transgene copy number (2 to 4 per cell). Cell viability of the final KYV-101 drug product ranged from 87–97%. KYV-101 displayed robust CD19-dependent and effector dose-related release of the pro-inflammatory cytokine IFN-γ. ConclusionsKYV-101 manufacturing yielded a CAR T-cell product with high viability and consistent composition and functionality, regardless of disease indication, pre-treatment, and heterogeneity of the incoming material. Cryopreservation of the apheresis and final drug product enabled widespread distribution. These results support the robustness of the manufacturing process for the fully human KYV-101 anti-CD19 CAR T-cell therapy drug product for patients across diverse autoimmune disease types.

The effect of the nucleolar protein ZNF385A on the ribosomal DNA copy number variation in response to Cr(VI)-induced DNA damage

Hexavalent chromium [Cr(VI)] is a widely distributed carcinogen in industrial contexts and general environmental contexts. Emerging research highlights the central role of ribosomal DNA (rDNA) in DNA Damage Responses (DDRs). However, there remains a lack of investigation into the potential dose-dependent relationship between exposure to Cr(VI) and alterations in rDNA copy number (CN), as well as the related mechanisms underlying these effects. A molecular epidemiological investigation involving 67 workers exposed to Cr(VI) and 75 unexposed controls was conducted. There was a notable increase in ZNF385A expression, variations in rDNA CN, and elevated γH2AX levels in the peripheral blood of Cr(VI)-exposed workers. Restricted cubic spline (RCS) models showed that blood Cr levels in the exposed population exhibited non-linear dose-dependent relationships with γH2AX, rDNA CN, and ZNF385A. Of considerable interest, there were robust and positive associations between ZNF385A and both γH2AX and rDNA CN. Further in vitro experiments provided concrete evidence that Cr(VI) simultaneously caused an increase in ZNF385A expression and variations in rDNA CN. ZNF385A-depleted cells showed increased sensitivity to Cr(VI)-mediated DDRs and alterations in rDNA CN. This study indicated that ZNF385A played a highly significant role in the rDNA CN variation in response to Cr(VI)-induced DNA damage.

Differences in activity and stability drive transposable element variation in tropical and temperate maize.

Much of the profound interspecific variation in genome content has been attributed to transposable elements (TEs). To explore the extent of TE variation within species, we developed an optimized open-source algorithm, panEDTA, to de novo annotate TEs in a pangenome context. We then generated a unified TE annotation for a maize pangenome derived from 26 reference-quality genomes, which reveals an excess of 35.1 Mb of TE sequences per genome in tropical maize relative to temperate maize. A small number (n = 216) of TE families, mainly LTR retrotransposons, drive these differences. Evidence from the methylome, transcriptome, LTR age distribution, and LTR insertional polymorphisms reveals that 64.7% of the variability is contributed by LTR families that are young, less methylated, and more expressed in tropical maize, whereas 18.5% is driven by LTR families with removal or loss in temperate maize. Additionally, we find enrichment for Young LTR families adjacent to nucleotide-binding and leucine-rich repeat (NLR) clusters of varying copy number across lines, suggesting TE activity may be associated with disease resistance in maize.

Evolution of extended-spectrum β-lactamase-producing ST131 Escherichia coli at a single hospital over 15 years

Escherichia coli multi-locus sequence type ST131 is a globally distributed pandemic lineage that causes multidrug-resistant extra-intestinal infections. ST131 E. coli frequently produce extended-spectrum β-lactamases (ESBLs), which confer resistance to many β-lactam antibiotics and make infections difficult to treat. We sequenced the genomes of 154 ESBL-producing E. coli clinical isolates belonging to the ST131 lineage from patients at the University of Pittsburgh Medical Center (UPMC) between 2004 and 2018. Isolates belonged to the well described ST131 clades A (8%), B (3%), and C (89%). Time-dated phylogenetic analysis estimated that the most recent common ancestor (MRCA) for all clade C isolates emerged around 1989, consistent with previous studies. We identified multiple genes potentially under selection in clade C, including the cell wall assembly gene ftsI, the LPS biosynthesis gene arnC, and the yersiniabactin uptake receptor fyuA. Diverse ESBL-encoding genes belonging to the blaCTX-M, blaSHV, and blaTEM families were identified; these genes were found at varying numbers of loci and in variable numbers of copies across isolates. Analysis of ESBL flanking regions revealed diverse mobile elements that varied by ESBL type. Overall, our findings show that ST131 subclade C dominated among patients and uncover possible signals of ongoing adaptation within this ST131 lineage.

EPAD1 Orthologs Play a Conserved Role in Pollen Exine Patterning.

The pollen wall protects pollen during dispersal and is critical for pollination recognition. In the Poaceae family, the pollen exine stereostructure exhibits a high degree of conservation with similar patterns across species. However, there remains controversy regarding the conservation of key factors involved in its formation among various Poaceae species. EPAD1, as a gene specific to the Poaceae family, and its orthologous genes play a conserved role in pollen wall formation in wheat and rice. However, they do not appear to have significant functions in maize. To further confirm the conserved function of EPAD1 in Poaceae, we performed an analysis on four EPAD1 orthologs from two distinct sub-clades within the Poaceae family. The two functional redundant barley EPAD1 genes (HvEPAD1 and HvEPAD2) from the BOP clade, along with the single copy of sorghum (SbEPAD1) and millet (SiEPAD1) from the PACMAD clade were examined. The CRISPR-Cas9-generated mutants all exhibited defects in pollen wall formation, consistent with previous findings on EPAD1 in rice and wheat. Interestingly, in barley, hvepad2 single mutant also showed apical spikelets abortion, aligning with a decreased expression level of HvEPAD1 and HvEPAD2 from the apical to the bottom of the spike. Our finding provides evidence that EPAD1 orthologs contribute to Poaceae specific pollen exine pattern formation via maintaining primexine integrity despite potential variations in copy numbers across different species.

Relative rDNA copy number is not associated with resistance training-induced skeletal muscle hypertrophy and does not affect myotube anabolism in vitro.

Ribosomal DNA (rDNA) copies exist across multiple chromosomes and inter-individual variation in copy number is speculated to influence the hypertrophic response to resistance training. Thus, we examined if rDNA copy number was associated with resistance training-induced skeletal muscle hypertrophy. Participants (n=53 males, 21±1 years old; n=29 females, 21±2 years old) performed 10-12 weeks of full-body resistance training. Hypertrophy outcomes were determined, as was relative rDNA copy number from pre-intervention vastus lateralis (VL) biopsies. Pre- and post-intervention VL biopsy total RNA was assayed in all participants, and mRNA/rRNA markers of ribosome content and biogenesis were also assayed in the 29 females prior to training, 24 hours following training bout 1, and in the basal state after 10 weeks of training. Across all participants, no significant associations were evident between relative rDNA copy number and training-induced changes in whole body lean mass (r = -0.034, p=0.764), vastus lateralis thickness (r = 0.093, p=0.408), mean myofiber cross-sectional area (r = -0.128, p=0.259), or changes in muscle RNA concentrations (r = 0.026, p=0.818), and these trends were similar when examining each gender. However, all Pol-I regulon mRNAs as well as 45S pre-rRNA, 28S rRNA and 18S rRNA increased 24 hours following the first training bout in females. Follow-up studies using LHCN-M2 myotubes demonstrated a reduction in relative rDNA copy number induced by bisphenol A (BPA) did not significantly affect insulin-like-growth factor-induced myotube hypertrophy. These findings suggest relative rDNA copy number is not associated with myofiber hypertrophy.

Ongoing genome doubling promotes evolvability and immune dysregulation in ovarian cancer.

Whole-genome doubling (WGD) is a critical driver of tumor development and is linked to drug resistance and metastasis in solid malignancies. Here, we demonstrate that WGD is an ongoing mutational process in tumor evolution. Using single-cell whole-genome sequencing, we measured and modeled how WGD events are distributed across cellular populations within tumors and associated WGD dynamics with properties of genome diversification and phenotypic consequences of innate immunity. We studied WGD evolution in 65 high-grade serous ovarian cancer (HGSOC) tissue samples from 40 patients, yielding 29,481 tumor cell genomes. We found near-ubiquitous evidence of WGD as an ongoing mutational process promoting cell-cell diversity, high rates of chromosomal missegregation, and consequent micronucleation. Using a novel mutation-based WGD timing method, doubleTime , we delineated specific modes by which WGD can drive tumor evolution: (i) unitary evolutionary origin followed by significant diversification, (ii) independent WGD events on a pre-existing background of copy number diversity, and (iii) evolutionarily late clonal expansions of WGD populations. Additionally, through integrated single-cell RNA sequencing and high-resolution immunofluorescence microscopy, we found that inflammatory signaling and cGAS-STING pathway activation result from ongoing chromosomal instability and are restricted to tumors that remain predominantly diploid. This contrasted with predominantly WGD tumors, which exhibited significant quiescent and immunosuppressive phenotypic states. Together, these findings establish WGD as an evolutionarily 'active' mutational process that promotes evolvability and dysregulated immunity in late stage ovarian cancer.

Dynamic Expansions and Retinal Expression of Spectrally Distinct Short-Wavelength Opsin Genes in Sea Snakes.

The photopigment-encoding visual opsin genes that mediate color perception show great variation in copy number and adaptive function across vertebrates. An open question is how this variation has been shaped by the interaction of lineage-specific structural genomic architecture and ecological selection pressures. We contribute to this issue by investigating the expansion dynamics and expression of the duplicated Short-Wavelength-Sensitive-1 opsin (SWS1) in sea snakes (Elapidae). We generated one new genome, 45 resequencing datasets, 10 retinal transcriptomes, and 81 SWS1 exon sequences for sea snakes, and analyzed these alongside 16 existing genomes for sea snakes and their terrestrial relatives. Our analyses revealed multiple independent transitions in SWS1 copy number in the marine Hydrophis clade, with at least three lineages having multiple intact SWS1 genes: the previously studied Hydrophis cyanocinctus and at least two close relatives of this species; Hydrophis atriceps and Hydrophis fasciatus; and an individual Hydrophis curtus. In each lineage, gene copy divergence at a key spectral tuning site resulted in distinct UV and Violet/Blue-sensitive SWS1 subtypes. Both spectral variants were simultaneously expressed in the retinae of H. cyanocinctus and H. atriceps, providing the first evidence that these SWS1 expansions confer novel phenotypes. Finally, chromosome annotation for nine species revealed shared structural features in proximity to SWS1 regardless of copy number. If these features are associated with SWS1 duplication, expanded opsin complements could be more common in snakes than is currently recognized. Alternatively, selection pressures specific to aquatic environments could favor improved chromatic distinction in just some lineages.

Transcriptional Silencing of 35S rDNA in Tragopogon porrifolius Correlates with Cytosine Methylation in Sequence-Specific Manner.

Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.