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American Journal of Botany Feb 2019Studies of gene expression and polyploidy are typically restricted to characterizing differences in transcript concentration. Using diploid and autotetraploid Tolmiea,... (Comparative Study)
Comparative Study
PREMISE OF THE STUDY
Studies of gene expression and polyploidy are typically restricted to characterizing differences in transcript concentration. Using diploid and autotetraploid Tolmiea, we present an integrated approach for cross-ploidy comparisons that account for differences in transcriptome size and cell density and make multiple comparisons of transcript abundance.
METHODS
We use RNA spike-in standards in concert with cell size and density to identify and correct for differences in transcriptome size and compare levels of gene expression across multiple scales: per transcriptome, per cell, and per biomass.
KEY RESULTS
In total, ~17% of all loci were identified as differentially expressed (DEGs) between the diploid and autopolyploid species. The per-transcriptome normalization, the method researchers typically use, captured the fewest DEGs (58% of total DEGs) and failed to detect any DEGs not found by the alternative normalizations. When transcript abundance was normalized per biomass and per cell, ~66% and ~82% of the total DEGs were recovered, respectively. The discrepancy between per-transcriptome and per-cell recovery of DEGs occurs because per-transcriptome normalizations are concentration-based and therefore blind to differences in transcriptome size.
CONCLUSIONS
While each normalization enables valid comparisons at biologically relevant scales, a holistic comparison of multiple normalizations provides additional explanatory power not available from any single approach. Notably, autotetraploid loci tend to conserve diploid-like transcript abundance per biomass through increased gene expression per cell, and these loci are enriched for photosynthesis-related functions.
Topics: Biomass; Cell Size; Diploidy; Gene Expression; Saxifragaceae; Tetraploidy; Transcriptome
PubMed: 30779448
DOI: 10.1002/ajb2.1239 -
MBio Aug 2022species are obligate fungal biotrophs that colonize the lungs of mammals. They cause deadly pneumonia in immunocompromised hosts. The sexual phase seems obligate during...
species are obligate fungal biotrophs that colonize the lungs of mammals. They cause deadly pneumonia in immunocompromised hosts. The sexual phase seems obligate during their life cycle and essential for survival because it is believed to ensure proliferation and transmission between hosts. Here, we consider if the sexual phase is initiated by the fusion of two cells or by nucleus duplication in order to generate diploid cells that can undergo meiosis. The juxtaposition of the nucleus-associated organelles of pairs of cells with fused cytoplasmic membranes demonstrated that cell fusion can occur. Nevertheless, the frequency of cell fusion remains to be determined, and it cannot be excluded that both cell fusion and nucleus duplication are used to ensure the occurrence of the essential sexual phase. culturing of these fungi is a major milestone that could clarify the issue.
Topics: Animals; Cell Fusion; Diploidy; Mammals; Meiosis; Pneumocystis; Reproduction
PubMed: 35726921
DOI: 10.1128/mbio.00859-22 -
G3 (Bethesda, Md.) Aug 2022The expression of nuclear and mitochondrial genes, as well as their coordinated control, regulates cell proliferation, individual development, and disease in animals....
The expression of nuclear and mitochondrial genes, as well as their coordinated control, regulates cell proliferation, individual development, and disease in animals. However, the potential coregulation between nuclear and mitochondrial genes is unclear in triploid fishes. The two triploids (R2C and RC2) with distinct mitochondrial genomes but similar nuclear genomes exhibit different embryonic development times and growth rates. They are an excellent model for studying how nuclear and mitochondrial genes coordinate. Here, we performed the mRNA-seq of four stages of embryonic development (blastula, gastrula, segmentation, and hatching periods) in the two triploids (R2C and RC2) and their diploid inbred parents (red crucian carp and common carp). After establishing the four patterns of mitochondrial and nuclear gene expression, 270 nuclear genes regulated by mitochondrial genes were predicted. The expression levels of APC16 and Trim33 were higher in RC2 than in R2C, suggesting their potential effects on regulating embryonic development time. In addition, 308 differentially expressed genes filtered from the list of nuclear-encoded mitochondrial genes described by Mercer et al. in 2011 were considered potential genes for which nuclear genes regulate mitochondrial function. The findings might aid in our understanding of the correlation between mitochondrial and nuclear genomes as well as their synergistic effects on embryonic development.
Topics: Animals; Carps; Cell Nucleus; Diploidy; Genes, Mitochondrial; Triploidy
PubMed: 35924985
DOI: 10.1093/g3journal/jkac197 -
Advanced Science (Weinheim,... Mar 2023Synthetic biology has been represented by the creation of artificial life forms at the genomic scale. In this work, a CRISPR-based chromosome-doubling technique is...
Synthetic biology has been represented by the creation of artificial life forms at the genomic scale. In this work, a CRISPR-based chromosome-doubling technique is designed to first construct an artificial diploid Escherichia coli cell. The stable single-cell diploid E. coli is isolated by both maximal dilution plating and flow cytometry, and confirmed with quantitative PCR, fluorescent in situ hybridization, and third-generation genome sequencing. The diploid E. coli has a greatly reduced growth rate and elongated cells at 4-5 µm. It is robust against radiation, and the survival rate after exposure to UV increased 40-fold relative to WT. As a novel life form, the artificial diploid E. coli is an ideal substrate for research fundamental questions in life science concerning polyploidy. And this technique may be applied to other bacteria.
Topics: Diploidy; Escherichia coli; In Situ Hybridization, Fluorescence; Polyploidy; Chromosomes, Plant
PubMed: 36642845
DOI: 10.1002/advs.202205855 -
American Journal of Clinical Pathology Jun 1972The use of both rhesus monkey kidney and human fetal diploid cell cultures for attempts to isolate virus from human clinical specimens recovered approximately 30% more... (Review)
Review
The use of both rhesus monkey kidney and human fetal diploid cell cultures for attempts to isolate virus from human clinical specimens recovered approximately 30% more isolates from fecal, throat, skin lesion, and tissue specimens than were recovered in a single cell culture system. The sensitivities of the two cell types for isolation of viruses in the major groups infecting man are compared. The use of both BS-C-1 and RK-13 cells yielded approximately 25% more rubella virus isolates than were recovered in a single cell system. Procedures such as the establishment of cell cultures from tissues suspected of harboring viruses, cocultivation of cells from biopsy or autopsy specimens with other cell types, and the use of organ cultures, have in certain situations resulted in greater sensitivity for recovery of viruses. Micro cell culture tests have been devised for virus identification and antibody assays; these are far more economical than conventional tests in terms of reagents, space requirements, and personnel time. Methods have been developed for the production of higher-titered viral serologic antigens in cell culture systems.
Topics: Adenoviridae; Animals; Antigens, Viral; Cell Line; Cells, Cultured; Culture Techniques; Diploidy; Enterovirus; Fetus; Herpesviridae; Humans; Kidney; Lung; Macaca; Neutralization Tests; Orthomyxoviridae; Paramyxoviridae; Reoviridae; Vaccinia virus; Virus Diseases; Viruses
PubMed: 4337372
DOI: 10.1093/ajcp/57.6.820 -
STAR Protocols Sep 2020Murine cardiomyocytes undergo proliferation, multinucleation, and polyploidization during the first 3 weeks of postnatal life, resulting in a mixture of diploid and...
Murine cardiomyocytes undergo proliferation, multinucleation, and polyploidization during the first 3 weeks of postnatal life, resulting in a mixture of diploid and tetraploid cardiomyocytes in the heart. Understanding the molecular differences between diploid and tetraploid cardiomyocytes from these processes has been limited due to lack of unique markers and their heterogenous origins. Here, we apply single-nucleus RNA-sequencing to fluorescence-activated cell sorting-selected diploid and tetraploid cardiomyocytes to characterize their heterogeneity and molecular distinctions. For complete details on the use and execution of this protocol, please refer to Cui et al. (2020).
Topics: Animals; Cell Culture Techniques; Cells, Cultured; Diploidy; Gene Expression Profiling; Mice; Myocytes, Cardiac; Single-Cell Analysis; Tetraploidy; Transcriptome
PubMed: 33111095
DOI: 10.1016/j.xpro.2020.100049 -
Human Vaccines & Immunotherapeutics 2019The large-scale production of a human diploid cell (HDC) vaccine (HDCV) for rabies is limited by several technical challenges. Kanghua Biological Products Co., Ltd., has... (Randomized Controlled Trial)
Randomized Controlled Trial
The large-scale production of a human diploid cell (HDC) vaccine (HDCV) for rabies is limited by several technical challenges. Kanghua Biological Products Co., Ltd., has successfully used microcarrier technology for the large-scale culture of HDCs in bioreactors to develop a lyophilized and purified HDCV. In this blinded, randomized, parallel-group study conducted between July and October 2014 in Mianzhu, Sichuan Province, China, we monitored the safety and immunogenicity of this vaccine in a healthy population vaccinated according to the Essen post-exposure immunization schedule. A hamster kidney cell vaccine was used as the control. Adverse reactions were monitored 0.5, 6, 24, 48, and 72 h post vaccination to assess safety. Neutralizing antibodies in venous blood were measured on day 7, 14, and 72 to evaluate the immunogenicity of the vaccine while follow-up monitoring continued for 1 month. No serious adverse reactions were observed in any volunteer. The incidence rates of systemic and local adverse reactions were, respectively, 10.6% and 2.9% in the test group and 20.0% and 13.6% in the control group. After the third injection, the positive conversion rates of antibodies in the test and control groups were 100% and 98.82%, respectively. In addition, the average antibody titers on day 7, 14, and 42, were respectively, 1.71, 2.72, and 1.29 times higher than those in the control group. These results indicate that HDCV had a better safety profile and higher immunogenicity than the hamster kidney cell rabies vaccine. Trial registration number: 20130602.
Topics: Adult; Animals; Antibodies, Neutralizing; Antibodies, Viral; Cell Culture Techniques; Cricetinae; Diploidy; Female; Freeze Drying; Humans; Immunization Schedule; Immunogenicity, Vaccine; Injections, Intramuscular; Kidney; Male; Microspheres; Middle Aged; Rabies; Rabies Vaccines; Rabies virus
PubMed: 30457436
DOI: 10.1080/21645515.2018.1549450 -
American Journal of Botany Feb 2022Endoreduplication, nonheritable duplication of a nuclear genome, is widespread in plants and plays a role in developmental processes related to cell differentiation....
PREMISE
Endoreduplication, nonheritable duplication of a nuclear genome, is widespread in plants and plays a role in developmental processes related to cell differentiation. However, neither ecological nor cytological factors influencing intraspecific variation in endoreduplication are fully understood.
METHODS
We cultivated plants covering the range-wide natural diversity of diploid and tetraploid populations of Arabidopsis arenosa in common conditions to investigate the effect of original ploidy level on endoreduplication. We also raised plants from several foothill and alpine populations from different lineages and of both ploidies to test for the effect of elevation. We determined the endoreduplication level in leaves of young plants by flow cytometry. Using RNA-seq data available for our populations, we analyzed gene expression analysis in individuals that differed in endoreduplication level.
RESULTS
We found intraspecific variation in endoreduplication that was mainly driven by the original ploidy level of populations, with significantly higher endoreduplication in diploids. An effect of elevation was also found within each ploidy, yet its direction exhibited rather regional-specific patterns. Transcriptomic analysis comparing individuals with high vs. low endopolyploidy revealed a majority of differentially expressed genes related to the stress and hormone response and to modifications especially in the cell wall and in chloroplasts.
CONCLUSIONS
Our results support the general assumption of higher potential of low-ploidy organisms to undergo endoreduplication and suggest that endoreduplication is further integrated within the stress response pathways for a fine-tune adjustment of the endoreduplication process to their local environment.
Topics: Arabidopsis; Diploidy; Endoreduplication; Ploidies; Tetraploidy
PubMed: 35137947
DOI: 10.1002/ajb2.1818 -
American Journal of Botany Dec 2020Hybridization plays a key role in introgressive adaptation, speciation, and adaptive radiation as a source of evolutionary innovation. Hybridization is considered common...
PREMISE
Hybridization plays a key role in introgressive adaptation, speciation, and adaptive radiation as a source of evolutionary innovation. Hybridization is considered common in Arctostaphylos, yet species boundaries are retained in stands containing multiple species. Arctostaphylos contains diploids and tetraploids, and recent phylogenies indicate two clades; we hypothesize combinations of these traits limit or promote hybridization rates.
METHODS
We statistically analyzed co-occurrence patterns of species by clade membership and ploidy level from 87 random 0.1 ha plots. We sampled multiple sites to analyze for percent hybridization based on morphology. Finally, phenophases were analyzed by scoring herbarium sheets for a large number of taxa from both clades as well as tetraploids, and second, surveying three field sites over two years for divergence in phenological stages between co-occurring taxa.
RESULTS
Most taxa in Arctostaphylos are allopatric relative to other congenerics. When two taxa co-occur, the patterns are a diploid with a tetraploid, or two diploids from different clades. When three taxa co-occur, the pattern is two diploids from different clades and a tetraploid. Field and herbarium data both indicate flowering phenology is displaced between diploids from the two clades; one of the diploid clades and tetraploids overlap considerably.
CONCLUSIONS
The two deep clades in Arctostaphylos are genetically distant, with hybrids rare or non-existent when taxa co-occur. Reproductive isolation between clades is enhanced by displaced flowering phenology for co-occurring species. Within clades, taxa appear to have few reproductive barriers other than an allopatric distribution or different ploidy levels.
Topics: Arctostaphylos; Diploidy; Hybridization, Genetic; Reproductive Isolation; Sympatry
PubMed: 33274449
DOI: 10.1002/ajb2.1576 -
Analytical Chemistry Oct 2017To advance an understanding of cellular regulation and function it is crucial to identify molecular contents in cellular organelles, which accommodate specific...
To advance an understanding of cellular regulation and function it is crucial to identify molecular contents in cellular organelles, which accommodate specific biochemical processes. Toward achievement of this goal, we applied micro-Raman-Biomolecular Component Analysis assay for molecular profiling of major organelles in live cells. We used this assay for comparative analysis of proteins 3D conformation and quantification of proteins, RNA, and lipids concentrations in nucleoli, endoplasmic reticulum, and mitochondria of WI 38 diploid lung fibroblasts and HeLa cancer cells. Obtained data show substantial differences in the concentrations and conformations of proteins in the studied organelles. Moreover, differences in the intraorganellar concentrations of RNA and lipids between these cell lines were found. We report the biological significance of obtained macromolecular profiles and advocate for micro-Raman BCA assay as a valuable proteomics tool.
Topics: Diploidy; Endoplasmic Reticulum; Fibroblasts; HeLa Cells; Humans; Lipids; Mitochondria; Proteins; RNA; Spectrum Analysis, Raman
PubMed: 28910082
DOI: 10.1021/acs.analchem.7b02822