scholarly journals PREIMPLANTATION GENETIC TESTING: Single-cell technologies at the forefront of PGT and embryo research

Reproduction ◽  
2020 ◽  
Vol 160 (5) ◽  
pp. A19-A31
Author(s):  
Olga Tšuiko ◽  
Elia Fernandez Gallardo ◽  
Thierry Voet ◽  
Joris Robert Vermeesch
Keyword(s):  
Single Cell ◽  
Embryo Development ◽  
Single Gene ◽  
Simultaneous Detection ◽  
Embryo Research ◽  
Chromosomal Mosaicism ◽  
Genetic Constitution ◽  
Aneuploidy Screening ◽  
Cell Technologies ◽  
The Impact

While chromosomal mosaicism in the embryo was observed already in the 1990s using both karyotyping and FISH technologies, the full extent of this phenomenon and the overall awareness of the consequences of chromosomal instability on embryo development has only come with the advent of sophisticated single-cell technologies. High-throughput techniques, such as DNA microarrays and massive parallel sequencing, have shifted single-cell genome research from evaluating a few loci at a time to the ability to perform comprehensive screening of all 24 chromosomes. The development of genome-wide single-cell haplotyping methods have also enabled for simultaneous detection of single-gene disorders and aneuploidy using a single universal protocol. Today, three decades later haplotyping-based embryo testing is performed worldwide to reliably detect virtually any Mendelian hereditary disease with a known cause, including autosomal-recessive, autosomal-dominant and X-linked disorders. At the same time, these single-cell assays have also provided unique insight into the complexity of embryo genome dynamics, by elucidating mechanistic origin, nature and developmental fate of embryonic aneuploidy. Understanding the impact of postzygotically acquired genomic aberrations on embryo development is essential to determine the still controversial diagnostic value of aneuploidy screening. For that reason, considerable efforts have been put into linking the genetic constitution of the embryo not only to its morphology and implantation potential, but more importantly to its transcriptome using single-cell RNA sequencing. Collectively, these breakthrough technologies have revolutionized single-cell research and clinical practice in assisted reproduction and led to unique discoveries in early embryogenesis.

scholarly journals SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells

2021 ◽  
Vol 7 (8) ◽  
pp. eabe3610
Author(s):  
Conor J. Kearney ◽  
Stephin J. Vervoort ◽  
Kelly M. Ramsbottom ◽  
Izabela Todorovski ◽  
Emily J. Lelliott ◽  
...  
Keyword(s):  
Single Cell ◽  
Posttranslational Modification ◽  
Cellular Differentiation ◽  
Single Cells ◽  
Simultaneous Detection ◽  
Surface Protein ◽  
Rna Seq ◽  
Cell Level ◽  
Simultaneous Integration ◽  
Unique Surface

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.

scholarly journals Morphodynamic signatures of MDA-MB-231 single cells and cell doublets undergoing invasion in confined microenvironments

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xingjian Zhang ◽  
Trevor Chan ◽  
Michael Mak
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Leading Edge ◽  
Cell Group ◽  
Collective State ◽  
Long Term Effects ◽  
Cell Metastasis ◽  
Geometric Confinement ◽  
Short And Long Term ◽  
The Impact

AbstractCancer cell metastasis is a major factor in cancer-related mortality. During the process of metastasis, cancer cells exhibit migratory phenotypes and invade through pores in the dense extracellular matrix. However, the characterization of morphological and subcellular features of cells in similar migratory phenotypes and the effects of geometric confinement on cell morphodynamics are not well understood. Here, we investigate the phenotypes of highly aggressive MDA-MB-231 cells in single cell and cell doublet (an initial and simplified collective state) forms in confined microenvironments. We group phenotypically similar single cells and cell doublets and characterize related morphological and subcellular features. We further detect two distinct migratory phenotypes, fluctuating and non-fluctuating, within the fast migrating single cell group. In addition, we demonstrate an increase in the number of protrusions formed at the leading edge of cells after invasion through geometric confinement. Finally, we track the short and long term effects of varied degrees of confinement on protrusion formation. Overall, our findings elucidate the underlying morphological and subcellular features associated with different single cell and cell doublet phenotypes and the impact of invasion through confined geometry on cell behavior.

scholarly journals Optimizing expression quantitative trait locus mapping workflows for single-cell studies

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Anna S. E. Cuomo ◽  
Giordano Alvari ◽  
Christina B. Azodi ◽  
Davis J. McCarthy ◽  
Marc Jan Bonder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Trait Locus ◽  
Single Cell ◽  
Quantitative Trait ◽  
Cell Types ◽  
Expression Quantitative Trait Locus ◽  
Eqtl Mapping ◽  
Trait Locus ◽  
The Impact

Abstract Background Single-cell RNA sequencing (scRNA-seq) has enabled the unbiased, high-throughput quantification of gene expression specific to cell types and states. With the cost of scRNA-seq decreasing and techniques for sample multiplexing improving, population-scale scRNA-seq, and thus single-cell expression quantitative trait locus (sc-eQTL) mapping, is increasingly feasible. Mapping of sc-eQTL provides additional resolution to study the regulatory role of common genetic variants on gene expression across a plethora of cell types and states and promises to improve our understanding of genetic regulation across tissues in both health and disease. Results While previously established methods for bulk eQTL mapping can, in principle, be applied to sc-eQTL mapping, there are a number of open questions about how best to process scRNA-seq data and adapt bulk methods to optimize sc-eQTL mapping. Here, we evaluate the role of different normalization and aggregation strategies, covariate adjustment techniques, and multiple testing correction methods to establish best practice guidelines. We use both real and simulated datasets across single-cell technologies to systematically assess the impact of these different statistical approaches. Conclusion We provide recommendations for future single-cell eQTL studies that can yield up to twice as many eQTL discoveries as default approaches ported from bulk studies.

scholarly journals Mitochondrial O-GlcNAc Transferase Interacts with and Modifies Many Proteins and Its Up-Regulation Affects Mitochondrial Function and Cellular Energy Homeostasis

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2956
Author(s):  
Paweł Jóźwiak ◽  
Piotr Ciesielski ◽  
Piotr K. Zakrzewski ◽  
Karolina Kozal ◽  
Joanna Oracz ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Energy Homeostasis ◽  
Glucose Sensor ◽  
Single Gene ◽  
Mitochondrial Proteins ◽  
Mitochondrial Activity ◽  
Ros Generation ◽  
Inner Mitochondrial Membrane ◽  
Glcnac Transferase ◽  
The Impact

O-GlcNAcylation is a cell glucose sensor. The addition of O-GlcNAc moieties to target protein is catalyzed by the O-Linked N-acetylglucosamine transferase (OGT). OGT is encoded by a single gene that yields differentially spliced OGT isoforms. One of them is targeted to mitochondria (mOGT). Although the impact of O-GlcNAcylation on cancer cells biology is well documented, mOGT’s role remains poorly investigated. We performed studies using breast cancer cells with up-regulated mOGT or its catalytic inactive mutant to identify proteins specifically modified by mOGT. Proteomic approaches included isolation of mOGT protein partners and O-GlcNAcylated proteins from mitochondria-enriched fraction followed by their analysis by mass spectrometry. Moreover, we analyzed the impact of mOGT dysregulation on mitochondrial activity and cellular metabolism using a variety of biochemical assays. We found that mitochondrial OGT expression is glucose-dependent. Elevated mOGT expression affected the mitochondrial transmembrane potential and increased intramitochondrial ROS generation. Moreover, mOGT up-regulation caused a decrease in cellular ATP level. We identified many mitochondrial proteins as mOGT substrates. Most of these proteins are localized in the mitochondrial matrix and the inner mitochondrial membrane and participate in mitochondrial respiration, fatty acid metabolism, transport, translation, apoptosis, and mtDNA processes. Our findings suggest that mOGT interacts with and modifies many mitochondrial proteins, and its dysregulation affects cellular bioenergetics and mitochondria function.

scholarly journals DNA Methylation and Intra-Clonal Heterogeneity: The Chronic Myeloid Leukemia Model

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3587
Author(s):  
Benjamin Lebecque ◽  
Céline Bourgne ◽  
Véronique Vidal ◽  
Marc G. Berger
Keyword(s):  
Dna Methylation ◽  
Chronic Myeloid Leukemia ◽  
Fusion Protein ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Single Gene ◽  
Clonal Heterogeneity ◽  
Sequencing Technologies ◽  
Genome Wide ◽  
The Impact

Chronic Myeloid Leukemia (CML) is a model to investigate the impact of tumor intra-clonal heterogeneity in personalized medicine. Indeed, tyrosine kinase inhibitors (TKIs) target the BCR-ABL fusion protein, which is considered the major CML driver. TKI use has highlighted the existence of intra-clonal heterogeneity, as indicated by the persistence of a minority subclone for several years despite the presence of the target fusion protein in all cells. Epigenetic modifications could partly explain this heterogeneity. This review summarizes the results of DNA methylation studies in CML. Next-generation sequencing technologies allowed for moving from single-gene to genome-wide analyses showing that methylation abnormalities are much more widespread in CML cells. These data showed that global hypomethylation is associated with hypermethylation of specific sites already at diagnosis in the early phase of CML. The BCR-ABL-independence of some methylation profile alterations and the recent demonstration of the initial intra-clonal DNA methylation heterogeneity suggests that some DNA methylation alterations may be biomarkers of TKI sensitivity/resistance and of disease progression risk. These results also open perspectives for understanding the epigenetic/genetic background of CML predisposition and for developing new therapeutic strategies.

scholarly journals Neurodegenerative diseases: a hotbed for splicing defects and the potential therapies

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Dunhui Li ◽  
Craig Stewart McIntosh ◽  
Frank Louis Mastaglia ◽  
Steve Donald Wilton ◽  
May Thandar Aung-Htut
Keyword(s):  
Alternative Splicing ◽  
Neurodegenerative Diseases ◽  
Messenger Rna ◽  
Muscular Atrophy ◽  
Single Gene ◽  
Synaptic Function ◽  
Coding Regions ◽  
Splicing Defects ◽  
The Impact ◽  
Splicing Patterns

AbstractPrecursor messenger RNA (pre-mRNA) splicing is a fundamental step in eukaryotic gene expression that systematically removes non-coding regions (introns) and ligates coding regions (exons) into a continuous message (mature mRNA). This process is highly regulated and can be highly flexible through a process known as alternative splicing, which allows for several transcripts to arise from a single gene, thereby greatly increasing genetic plasticity and the diversity of proteome. Alternative splicing is particularly prevalent in neuronal cells, where the splicing patterns are continuously changing to maintain cellular homeostasis and promote neurogenesis, migration and synaptic function. The continuous changes in splicing patterns and a high demand on many cis- and trans-splicing factors contribute to the susceptibility of neuronal tissues to splicing defects. The resultant neurodegenerative diseases are a large group of disorders defined by a gradual loss of neurons and a progressive impairment in neuronal function. Several of the most common neurodegenerative diseases involve some form of splicing defect(s), such as Alzheimer’s disease, Parkinson’s disease and spinal muscular atrophy. Our growing understanding of RNA splicing has led to the explosion of research in the field of splice-switching antisense oligonucleotide therapeutics. Here we review our current understanding of the effects alternative splicing has on neuronal differentiation, neuronal migration, synaptic maturation and regulation, as well as the impact on neurodegenerative diseases. We will also review the current landscape of splice-switching antisense oligonucleotides as a therapeutic strategy for a number of common neurodegenerative disorders.

scholarly journals Prediction model for aneuploidy in early human embryo development revealed by single-cell analysis

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Maria Vera-Rodriguez ◽  
Shawn L. Chavez ◽  
Carmen Rubio ◽  
Renee A. Reijo Pera ◽  
Carlos Simon
Keyword(s):  
Prediction Model ◽  
Single Cell ◽  
Embryo Development ◽  
Human Embryo ◽  
Single Cell Analysis ◽  
Cell Analysis ◽  
Human Embryo Development ◽  
Early Human

Bridging scales: From cell biology to physiology using in situ single-cell technologies

Cell Systems ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 388-400
Author(s):  
Maeve P. Nagle ◽  
Gabriela S. Tam ◽  
Evan Maltz ◽  
Zachary Hemminger ◽  
Roy Wollman
Keyword(s):  
Single Cell ◽  
Cell Biology ◽  
Cell Technologies
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