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  • MinireviewMarch 31, 2021

    2 162 907

    Single-Cell Toolkits Opening a New Era for Cell Engineering

    Sean Lee , Jireh Kim , and Jong-Eun Park

    Mol. Cells 2021; 44(3): 127-135 https://doi.org/10.14348/molcells.2021.0002
    Abstract

    Abstract : Since the introduction of RNA sequencing (RNA-seq) as a high-throughput mRNA expression analysis tool, this procedure has been increasingly implemented to identify cell-level transcriptome changes in a myriad of model systems. However, early methods processed cell samples in bulk, and therefore the unique transcriptomic patterns of individual cells would be lost due to data averaging. Nonetheless, the recent and continuous development of new single-cell RNA sequencing (scRNA-seq) toolkits has enabled researchers to compare transcriptomes at a single-cell resolution, thus facilitating the analysis of individual cellular features and a deeper understanding of cellular functions. Nonetheless, the rapid evolution of high throughput single-cell “omics” tools has created the need for effective hypothesis verification strategies. Particularly, this issue could be addressed by coupling cell engineering techniques with single-cell sequencing. This approach has been successfully employed to gain further insights into disease pathogenesis and the dynamics of differentiation trajectories. Therefore, this review will discuss the current status of cell engineering toolkits and their contributions to single-cell and genome-wide data collection and analyses.

  • MinireviewMarch 31, 2021

    2 98 654
    Abstract

    Abstract : Senescent cells that gradually accumulate during aging are one of the leading causes of aging. While senolytics can improve aging in humans as well as mice by specifically eliminating senescent cells, the effect of the senolytics varies in different cell types, suggesting variations in senescence. Various factors can induce cellular senescence, and the rate of accumulation of senescent cells differ depending on the organ. In addition, since the heterogeneity is due to the spatiotemporal context of senescent cells, in vivo studies are needed to increase the understanding of senescent cells. Since current methods are often unable to distinguish senescent cells from other cells, efforts are being made to find markers commonly expressed in senescent cells using bulk RNA-sequencing. Moreover, single-cell RNA (scRNA) sequencing, which analyzes the transcripts of each cell, has been utilized to understand the in vivo characteristics of the rare senescent cells. Recently, transcriptomic cell atlases for each organ using this technology have been published in various species. Novel senescent cells that do not express previously established marker genes have been discovered in some organs. However, there is still insufficient information on senescent cells due to the limited throughput of the scRNA sequencing technology. Therefore, it is necessary to improve the throughput of the scRNA sequencing technology or develop a way to enrich the rare senescent cells. The in vivo senescent cell atlas that is established using rapidly developing single-cell technologies will contribute to the precise rejuvenation by specifically removing senescent cells in each tissue and individual.

  • Research ArticleMarch 31, 2021

    2 103 624

    UHRF1 Induces Methylation of the TXNIP Promoter and Down-Regulates Gene Expression in Cervical Cancer

    Min Jun Kim , Han Ju Lee , Mee Young Choi , Sang Soo Kang , Yoon Sook Kim , Jeong Kyu Shin , and Wan Sung Choi

    Mol. Cells 2021; 44(3): 146-159 https://doi.org/10.14348/molcells.2021.0001
    Abstract

    Abstract : DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.

  • Research ArticleMarch 31, 2021

    0 82 204

    Morphological Characterization of small, dumpy, and long Phenotypes in Caenorhabditis elegans

    Joshua Young Cho , Tae-Woo Choi , Seung Hyun Kim , Joohong Ahnn , and Sun-Kyung Lee

    Mol. Cells 2021; 44(3): 160-167 https://doi.org/10.14348/molcells.2021.2236
    Abstract

    Abstract : The determinant factors of an organism’s size during animal development have been explored from various angles but remain partially understood. In Caenorhabditis elegans, many genes affecting cuticle structure, cell growth, and proliferation have been identified to regulate the worm’s overall morphology, including body size. While various mutations in those genes directly result in changes in the morphological phenotypes, there is still a need for established, clear, and distinct standards to determine the apparent abnormality in a worm’s size and shape. In this study, we measured the body length, body width, terminal bulb length, and head size of mutant worms with reported Dumpy (Dpy), Small (Sma) or Long (Lon) phenotypes by plotting and comparing their respective ratios of various parameters. These results show that the Sma phenotypes are proportionally smaller overall with mild stoutness, and Dpy phenotypes are significantly stouter and have disproportionally small head size. This study provides a standard platform for determining morphological phenotypes designating and annotating mutants that exhibit body shape variations, defining the morphological phenotype of previously unexamined mutants.

  • Research ArticleMarch 31, 2021

    1 49 377

    Tsg101 Is Necessary for the Establishment and Maintenance of Mouse Retinal Pigment Epithelial Cell Polarity

    Dai Le , Soyeon Lim , Kwang Wook Min , Joon Woo Park , Youjoung Kim , Taejeong Ha , Kyeong Hwan Moon , Kay-Uwe Wagner , and Jin Woo Kim

    Mol. Cells 2021; 44(3): 168-178 https://doi.org/10.14348/molcells.2021.0027
    Abstract

    Abstract : The retinal pigment epithelium (RPE) forms a monolayer sheet separating the retina and choroid in vertebrate eyes. The polarized nature of RPE is maintained by distributing membrane proteins differentially along apico-basal axis. We found the distributions of these proteins differ in embryonic, post-natal, and mature mouse RPE, suggesting developmental regulation of protein trafficking. Thus, we deleted tumor susceptibility gene 101 (Tsg101), a key component of endosomal sorting complexes required for transport (ESCRT), in embryonic and mature RPE to determine whether ESCRT-mediated endocytic protein trafficking correlated with the establishment and maintenance of RPE polarity. Loss of Tsg101 severely disturbed the polarity of RPE, which forms irregular aggregates exhibiting non-polarized distribution of cell adhesion proteins and activation of epidermal growth factor receptor signaling. These findings suggest that ESCRT-mediated protein trafficking is essential for the development and maintenance of RPE cell polarity.

  • Research ArticleMarch 31, 2021

    0 52 239
    Abstract

    Abstract : Vancomycin response regulator (VncR) is a pneumococcal response regulator of the VncRS two-component signal transduction system (TCS) of Streptococcus pneumoniae. VncRS regulates bacterial autolysis and vancomycin resistance. VncR contains two different functional domains, the N-terminal receiver domain and C-terminal effector domain. Here, we investigated VncR C-terminal DNA binding domain (VncRc) structure using a crystallization approach. Crystallization was performed using the micro-batch method. The crystals diffracted to a 1.964 Å resolution and belonged to space group P212121. The crystal unit-cell parameters were a = 25.71 Å, b = 52.97 Å, and c = 60.61 Å. The structure of VncRc had a helix-turn-helix motif highly similar to the response regulator PhoB of Escherichia coli. In isothermal titration calorimetry and size exclusion chromatography results, VncR formed a complex with VncS, a sensor histidine kinase of pneumococcal TCS. Determination of VncR structure will provide insight into the mechanism by how VncR binds to target genes.

Mol. Cells
Nov 30, 2021 Vol.44 No.11
COVER PICTURE
3D quantitative images of the vesicular structure and the nucleolus using label free optical diffraction tomography (Kim et al., pp. 851-860).

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