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An improved reference of the grapevine genome reasserts the origin of the PN40024 highly homozygous genotype
Full text linkThe genome sequence of the diploid and highly homozygous Vitis vinifera genotype PN40024 serves as the reference for many grapevine studies. Despite several improvements to the PN40024 genome assembly, its current version PN12X.v2 is quite fragmented and only represents the haploid state of the genome with mixed haplotypes. In fact, being nearly homozygous, this genome contains several heterozygous regions that are yet to be resolved. Taking the opportunity of improvements that long-read sequencing technologies offer to fully discriminate haplotype sequences, an improved version of the reference, called PN40024.v4, was generated. Through incorporating long genomic sequencing reads to the assembly, the continuity of the 12X.v2 scaffolds was highly increased with a total number decreasing from 2,059 to 640 and a reduction in N bases of 88%. Additionally, the full alternative haplotype sequence was built for the first time, the chromosome anchoring was improved and the number of unplaced scaffolds was reduced by half. To obtain a high-quality gene annotation that outperforms previous versions, a liftover approach was complemented with an optimized annotation workflow for Vitis. Integration of the gene reference catalogue and its manual curation have also assisted in improving the annotation, while defining the most reliable estimation of 35,230 genes to date. Finally, we demonstrated that PN40024 resulted from 9 selfings of cv. "Helfensteiner" (cross of cv. "Pinot noir" and "Schiava grossa") instead of a single "Pinot noir". These advances will help maintain the PN40024 genome as a gold-standard reference, also contributing toward the eventual elaboration of the grapevine pangenome
Establishment and Culture of Patient-Derived Breast Organoids
No full textBreast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures
Long non-coding RNAs: definitions, functions, challenges and recommendations
No full textGenes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease
NETworking with cancer: The bidirectional interplay between cancer and neutrophil extracellular traps
No full textNeutrophils are major effectors and regulators of the immune system. They play critical roles not only in the eradication of pathogens but also in cancer initiation and progression. Conversely, the presence of cancer affects neutrophil activity, maturation, and lifespan. By promoting or repressing key neutrophil functions, cancer cells co-opt neutrophil biology to their advantage. This co-opting includes hijacking one of neutrophils' most striking pathogen defense mechanisms: the formation of neutrophil extracellular traps (NETs). NETs are web-like filamentous extracellular structures of DNA, histones, and cytotoxic granule-derived proteins. Here, we discuss the bidirectional interplay by which cancer stimulates NET formation, and NETs in turn support disease progression. We review how vascular dysfunction and thrombosis caused by neutrophils and NETs underlie an elevated risk of death from cardiovascular events in cancer patients. Finally, we propose therapeutic strategies that may be effective in targeting NETs in the clinical setting
A shape-shifting nuclease unravels structured RNA
Full text linkRNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts. Dis3-like 2 (Dis3L2) is a 3'-5' exoribonuclease that plays a critical role in human development. Dis3L2 independently degrades structured substrates, including coding and noncoding 3' uridylated RNAs. While the basis for Dis3L2's substrate recognition has been well characterized, the mechanism of structured RNA degradation by this family of enzymes is unknown. We characterized the discrete steps of the degradation cycle by determining cryogenic electron microscopy structures representing snapshots along the RNA turnover pathway and measuring kinetic parameters for RNA processing. We discovered a dramatic conformational change that is triggered by double-stranded RNA (dsRNA), repositioning two cold shock domains by 70 Å. This movement exposes a trihelix linker region, which acts as a wedge to separate the two RNA strands. Furthermore, we show that the trihelix linker is critical for dsRNA, but not single-stranded RNA, degradation. These findings reveal the conformational plasticity of Dis3L2 and detail a mechanism of structured RNA degradation
Deep photoautotrophic prokaryotes contribute substantially to carbon dynamics in oxygen-deficient waters in a permanently redox-stratified freshwater lake
No full textIn lakes, seasonal phytoplankton blooms and allochthonous plant debris intensify particulate organic carbon fluxes to the lakebed. Microbes associated with these particles likely vary with organic substrate lability and redox conditions. To explore microbial compositional responses to these variables, we analyzed particle-associated and free-living assemblages in the permanently redox-stratified Fayetteville Green Lake using 16 S rRNA amplicon sequencing during the peak and end of cyanobacterial and photoautotrophic sulfur bacterial blooms. Assemblage compositions were strongly influenced by redox conditions and particle association. Assemblage compositions varied seasonally above the lower oxycline boundary (summer—generalist heterotrophs; autumn—iron reducers and specialist heterotrophs), but not in the anoxic region below. Particle-associated assemblages were less diverse than free-living assemblages and were dominated by heterotrophs that putatively metabolize complex organic substrates, purple sulfur bacteria, sulfur-cycling Desulfocapsa, and eukaryotic algae. The least diverse particle-associated assemblages occurred near the lower oxycline boundary, where microbial activities and abundances were highest, and anoxygenic photoautotrophs were enriched. The low-diversity particle-associated heterotrophs likely remineralize complex organic substrates, releasing simpler organic substrates to free-living assemblages during transit, thereby influencing surrounding microbial diversity and function. Our results challenge the paradigm that phytoplankton from the shallow photic zone are the primary contributor to the vertical flux. We suggest that photoautotrophic prokaryotes from the deep photic zone contribute significantly to deep-water carbon in this environment, and possibly in other oxygen-deficient waters with sulfidic photic zones. Furthermore, results suggest that seasonally variable terrestrial carbon and metal inputs also influence microbial diversity and function in similar systems
eDNA Analysis of Goat-Grazed Rhamnus cathartica Soil Microbial Communities.
No full textBiological invasions of exotic species pose a significant threat to local biodiversity and ecosystem services. Redistribution of plant species in particular disrupts key structure-function relationships in the soil by altering the composition of microbial taxa. This study combines environmental DNA analysis with a goat-grazing intervention to investigate the taxonomic structure of soil microbiomes interacting with the invasive plant, Rhamnus cathartica (common Buckthorn). 16S (515F/806R) metabarcodes were amplified from soil DNA harvested before and after a goat-grazing regimen on buckthorn-invaded and control plots. Samples were sequenced on an Illumina iSeq, then analyzed using the DNA Subway Purple Line (QIIME 2.0) to process sequencing reads and calculate diversity indices. PCoA visualizations of Jaccard Beta diversity patterns revealed differences in community composition between buckthorn and control soils, but no effects from goat grazing. Five dominant taxa were identified in no-buckthorn control soils, accounting for 35 +/- 9.6 SD% of the average relative abundance of total sequences. Genus DA101 notably demonstrated the largest decrease in average relative abundance in the buckthorn and goat-grazed soils (% decrease = 6.4 +/- 2 SD), whereas the evenness of other taxa increased. Overall, buckthorn soil showed significant increases in Pielou's taxonomical evenness (Kruskal-Wallis H(2, N=29) = 10.9; p=0.004), but goat grazing had no impact on this score (p>0.01). Faith's phylogenetic diversity scores showed no significant differences between buckthorn, goat-grazed, and control soils (Kruskal-Wallis H(2, N=31) = 2.2; p=0.32). Taken together, these preliminary results illustrate that buckthorn increases taxonomical evenness but has no effect on overall phylogenetic diversity of soil communities. Moreover, buckthorn goat-grazing had little impact on soil community structure. Future studies will investigate the role of DA101 on buckthorn suppression as a possible mitigation strategy for this invasive species
Complete Sequence of a 641-kb Insertion of Mitochondrial DNA in the Arabidopsis thaliana Nuclear Genome
Full text linkIntracellular transfers of mitochondrial DNA continue to shape nuclear genomes. Chromosome 2 of the model plant Arabidopsis thaliana contains one of the largest known nuclear insertions of mitochondrial DNA (numts). Estimated at over 600 kb in size, this numt is larger than the entire Arabidopsis mitochondrial genome. The primary Arabidopsis nuclear reference genome contains less than half of the numt because of its structural complexity and repetitiveness. Recent data sets generated with improved long-read sequencing technologies (PacBio HiFi) provide an opportunity to finally determine the accurate sequence and structure of this numt. We performed a de novo assembly using sequencing data from recent initiatives to span the Arabidopsis centromeres, producing a gap-free sequence of the Chromosome 2 numt, which is 641 kb in length and has 99.933% nucleotide sequence identity with the actual mitochondrial genome. The numt assembly is consistent with the repetitive structure previously predicted from fiber-based fluorescent in situ hybridization. Nanopore sequencing data indicate that the numt has high levels of cytosine methylation, helping to explain its biased spectrum of nucleotide sequence divergence and supporting previous inferences that it is transcriptionally inactive. The original numt insertion appears to have involved multiple mitochondrial DNA copies with alternative structures that subsequently underwent an additional duplication event within the nuclear genome. This work provides insights into numt evolution, addresses one of the last unresolved regions of the Arabidopsis reference genome, and represents a resource for distinguishing between highly similar numt and mitochondrial sequences in studies of transcription, epigenetic modifications, and de novo mutations