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  1. Article ; Online: CRISPR/Cas9-Mediated Gene Knockout in Cells and Tissues Using Lentivirus.

    Lu, Jiaoyang / Wang, Shaohe

    Current protocols

    2023  Volume 3, Issue 5, Page(s) e772

    Abstract: CRISPR-Cas9 has become a powerful and popular gene editing tool. However, successful application of this tool in the lab can still be quite daunting to many newcomers to molecular biology, mostly because it is a relatively lengthy process involving ... ...

    Abstract CRISPR-Cas9 has become a powerful and popular gene editing tool. However, successful application of this tool in the lab can still be quite daunting to many newcomers to molecular biology, mostly because it is a relatively lengthy process involving multiple steps with variations of each step. Here, we provide a reliable, stepwise, and newcomer-friendly protocol to knock out a target gene in wild-type human fibroblasts. This protocol involves sgRNA design using CRISPOR, construction of an "all-in-one" vector expressing both sgRNA and Cas9 using Golden Gate cloning, streamlined production of high-titer lentiviruses in 1 week after molecular cloning, and transduction of cells to generate a knockout cell pool. We further introduce a protocol for lentiviral transduction of ex vivo mouse embryonic salivary epithelial explants. In summary, our protocol is useful for new researchers to apply CRISPR-Cas9 to generate stable gene knockout cells and tissue explants using lentivirus. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: sgRNA design Basic Protocol 2: Cloning sgRNA in plasmid vector containing Cas9 encoding sequence using golden gate cloning Basic Protocol 3: Lentivirus packaging Basic Protocol 4: Lentivirus transduction of cells Basic Protocol 5: Lentivirus transduction of salivary gland epithelial buds.
    MeSH term(s) Humans ; Animals ; Mice ; Mice, Knockout ; Lentivirus/genetics ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Cloning, Molecular ; Craniocerebral Trauma
    Language English
    Publishing date 2023-05-24
    Publishing country United States
    Document type Journal Article
    ISSN 2691-1299
    ISSN (online) 2691-1299
    DOI 10.1002/cpz1.772
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  2. Article ; Online: Tissue Morphogenesis Through Dynamic Cell and Matrix Interactions.

    Wu, Di / Yamada, Kenneth M / Wang, Shaohe

    Annual review of cell and developmental biology

    2023  Volume 39, Page(s) 123–144

    Abstract: Multicellular organisms generate tissues of diverse shapes and functions from cells and extracellular matrices. Their adhesion molecules mediate cell-cell and cell-matrix interactions, which not only play crucial roles in maintaining tissue integrity but ...

    Abstract Multicellular organisms generate tissues of diverse shapes and functions from cells and extracellular matrices. Their adhesion molecules mediate cell-cell and cell-matrix interactions, which not only play crucial roles in maintaining tissue integrity but also serve as key regulators of tissue morphogenesis. Cells constantly probe their environment to make decisions: They integrate chemical and mechanical information from the environment via diffusible ligand- or adhesion-based signaling to decide whether to release specific signaling molecules or enzymes, to divide or differentiate, to move away or stay, or even whether to live or die. These decisions in turn modify their environment, including the chemical nature and mechanical properties of the extracellular matrix. Tissue morphology is the physical manifestation of the remodeling of cells and matrices by their historical biochemical and biophysical landscapes. We review our understanding of matrix and adhesion molecules in tissue morphogenesis, with an emphasis on key physical interactions that drive morphogenesis.
    Language English
    Publishing date 2023-06-14
    Publishing country United States
    Document type Journal Article ; Review
    ZDB-ID 1293750-2
    ISSN 1530-8995 ; 1081-0706
    ISSN (online) 1530-8995
    ISSN 1081-0706
    DOI 10.1146/annurev-cellbio-020223-031019
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  3. Article ; Online: Single Molecule RNA FISH (smFISH) in Whole-Mount Mouse Embryonic Organs.

    Wang, Shaohe

    Current protocols in cell biology

    2018  Volume 83, Issue 1, Page(s) e79

    Abstract: Single molecule RNA fluorescence in situ hybridization (smFISH) has become the standard tool for high spatial resolution analysis of gene expression in the context of tissue organization. This article describes protocols to perform smFISH on whole-mount ... ...

    Abstract Single molecule RNA fluorescence in situ hybridization (smFISH) has become the standard tool for high spatial resolution analysis of gene expression in the context of tissue organization. This article describes protocols to perform smFISH on whole-mount mouse embryonic organs, where tissue organization can be compared to RNA expression by co-immunostaining of known protein markers. An enzymatic labeling strategy is also introduced to produce low-cost smFISH probes. Important considerations and practical guidelines for imaging smFISH samples using fluorescence confocal microscopy are described. Finally, a suite of custom-written ImageJ macros is included with detailed instructions to enable semi-automated smFISH image analysis of both 2D and 3D images. © 2018 by John Wiley & Sons, Inc.
    MeSH term(s) Animals ; Embryo, Mammalian ; Gene Expression Profiling/methods ; In Situ Hybridization, Fluorescence/methods ; Mice ; RNA/analysis ; Single Molecule Imaging
    Chemical Substances RNA (63231-63-0)
    Language English
    Publishing date 2018-11-05
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Intramural
    ISSN 1934-2616
    ISSN (online) 1934-2616
    DOI 10.1002/cpcb.79
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  4. Article ; Online: The kinase ZYG-1 phosphorylates the cartwheel protein SAS-5 to drive centriole assembly in C. elegans.

    Sankaralingam, Prabhu / Wang, Shaohe / Liu, Yan / Oegema, Karen F / O'Connell, Kevin F

    EMBO reports

    2024  

    Abstract: Centrioles organize centrosomes, the cell's primary microtubule-organizing centers (MTOCs). Centrioles double in number each cell cycle, and mis-regulation of this process is linked to diseases such as cancer and microcephaly. In C. elegans, centriole ... ...

    Abstract Centrioles organize centrosomes, the cell's primary microtubule-organizing centers (MTOCs). Centrioles double in number each cell cycle, and mis-regulation of this process is linked to diseases such as cancer and microcephaly. In C. elegans, centriole assembly is controlled by the Plk4 related-kinase ZYG-1, which recruits the SAS-5-SAS-6 complex. While the kinase activity of ZYG-1 is required for centriole assembly, how it functions has not been established. Here we report that ZYG-1 physically interacts with and phosphorylates SAS-5 on 17 conserved serine and threonine residues in vitro. Mutational scanning reveals that serine 10 and serines 331/338/340 are indispensable for proper centriole assembly. Embryos expressing SAS-5
    Language English
    Publishing date 2024-05-14
    Publishing country England
    Document type Journal Article
    ZDB-ID 2020896-0
    ISSN 1469-3178 ; 1469-221X
    ISSN (online) 1469-3178
    ISSN 1469-221X
    DOI 10.1038/s44319-024-00157-y
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  5. Article ; Online: A Comparative Study on the Properties of Rosin-Based Epoxy Resins with Different Flexible Chains

    Lianli Deng / Zehua Wang / Bailu Qu / Ying Liu / Wei Qiu / Shaohe Qi

    Polymers, Vol 15, Iss 21, p

    2023  Volume 4246

    Abstract: This study aims to reveal the effects of flexible chain lengths on rosin-based epoxy resin’s properties. Two rosin-based epoxy monomers with varying chain lengths were synthesized: AR-EGDE (derived from ethylene glycol diglycidyl ether-modified acrylic ... ...

    Abstract This study aims to reveal the effects of flexible chain lengths on rosin-based epoxy resin’s properties. Two rosin-based epoxy monomers with varying chain lengths were synthesized: AR-EGDE (derived from ethylene glycol diglycidyl ether-modified acrylic acid rosin) and ARE (derived from acrylic acid rosin and epichlorohydrin). Diethylenetriamine (DETA), triethylenetetramine (TETA), and tetraethylenepentamine (TEPA) with different flexible chain lengths were used as curing agents. The adhesion, impact, pencil hardness, flexibility, water and heat resistance, and weatherability of the epoxy resins were systematically examined. It was found that when the flexible chains of rosin-based epoxy monomers were grown from ARE to AR-EGDE, due to the increased space of rosin-based fused rings, the toughness, adhesion, and water resistance of the rosin-based epoxy resins were enhanced, while the pencil hardness and heat resistance decreased. However, when the flexible chains of curing agents were lengthened, the resin’s performance did not change significantly because the space between the fused rings changed little. This indicates that the properties of the rosin-based resins can only be altered when the introduced flexible chain increases the space between the fused rings. The study also compared rosin-based resins to E20, a commercial petroleum-based epoxy of the bisphenol A type. The rosin-based resins demonstrated superior adhesion, water resistance, and weatherability compared to the E20 resins, indicating the remarkable durability of the rosin-based resin.
    Keywords rosin ; flexible chain ; film property ; bio-based epoxy ; curing agent ; Organic chemistry ; QD241-441
    Subject code 910
    Language English
    Publishing date 2023-10-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article: Fractal study on the dynamic fracture of red sandstone after F-T cycles

    Liu, Shi / Xu, Jinyu / Liu, Shaohe / Wang, Peng

    Environmental earth sciences. 2022 Mar., v. 81, no. 5

    2022  

    Abstract: Rock is an easily weathered material. Especially in alpine regions, freeze–thaw (F-T) cycles have a significant effect on the mechanical behavior of rock. A dynamic disturbance load further complicates the problem. Rock is also a multi-scale material; it ...

    Abstract Rock is an easily weathered material. Especially in alpine regions, freeze–thaw (F-T) cycles have a significant effect on the mechanical behavior of rock. A dynamic disturbance load further complicates the problem. Rock is also a multi-scale material; it may be more suitable to investigate the change in the macroscopic behavior from a microscopic perspective. Since rock fractures exhibit self-similarity, the material responses can be analyzed using fractal theory to determine the chaotic morphology of macroscopic fractures. We use a Φ 100 mm split Hopkinson pressure bar (SHPB) to perform dynamic tests on red sandstone after F-T cycles at three impact strength levels to obtain the dynamic fracture morphology. The fractal dimension of the dynamic fracture is calculated. The macroscopic fracture degree is quantitatively characterized. The relationship between the fractal dimension and the F-T cycles and impact strength are established. A scanning electron microscope (SEM) is used to obtain microscopic images of the dynamic fracture, which are processed to extract quantitative information, including the number, area, length, and width of the pores. The relationship between the fractal dimension and the pore information is investigated to reflect the macro-microscopic characteristics of rock under the coupling environment of F-T cycles and impact loads.
    Keywords fractal dimensions ; freeze-thaw cycles ; impact strength ; sandstone
    Language English
    Dates of publication 2022-03
    Size p. 152.
    Publishing place Springer Berlin Heidelberg
    Document type Article
    ZDB-ID 2493699-6
    ISSN 1866-6299 ; 1866-6280
    ISSN (online) 1866-6299
    ISSN 1866-6280
    DOI 10.1007/s12665-022-10270-7
    Database NAL-Catalogue (AGRICOLA)

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  7. Article ; Online: A Comparative Study on the Properties of Rosin-Based Epoxy Resins with Different Flexible Chains.

    Deng, Lianli / Wang, Zehua / Qu, Bailu / Liu, Ying / Qiu, Wei / Qi, Shaohe

    Polymers

    2023  Volume 15, Issue 21

    Abstract: This study aims to reveal the effects of flexible chain lengths on rosin-based epoxy resin's properties. Two rosin-based epoxy monomers with varying chain lengths were synthesized: AR-EGDE (derived from ethylene glycol diglycidyl ether-modified acrylic ... ...

    Abstract This study aims to reveal the effects of flexible chain lengths on rosin-based epoxy resin's properties. Two rosin-based epoxy monomers with varying chain lengths were synthesized: AR-EGDE (derived from ethylene glycol diglycidyl ether-modified acrylic acid rosin) and ARE (derived from acrylic acid rosin and epichlorohydrin). Diethylenetriamine (DETA), triethylenetetramine (TETA), and tetraethylenepentamine (TEPA) with different flexible chain lengths were used as curing agents. The adhesion, impact, pencil hardness, flexibility, water and heat resistance, and weatherability of the epoxy resins were systematically examined. It was found that when the flexible chains of rosin-based epoxy monomers were grown from ARE to AR-EGDE, due to the increased space of rosin-based fused rings, the toughness, adhesion, and water resistance of the rosin-based epoxy resins were enhanced, while the pencil hardness and heat resistance decreased. However, when the flexible chains of curing agents were lengthened, the resin's performance did not change significantly because the space between the fused rings changed little. This indicates that the properties of the rosin-based resins can only be altered when the introduced flexible chain increases the space between the fused rings. The study also compared rosin-based resins to E20, a commercial petroleum-based epoxy of the bisphenol A type. The rosin-based resins demonstrated superior adhesion, water resistance, and weatherability compared to the E20 resins, indicating the remarkable durability of the rosin-based resin.
    Language English
    Publishing date 2023-10-28
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2527146-5
    ISSN 2073-4360 ; 2073-4360
    ISSN (online) 2073-4360
    ISSN 2073-4360
    DOI 10.3390/polym15214246
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  8. Article ; Online: Microtubule-dependent apical polarization of basement membrane matrix mRNAs in mouse epithelial cells.

    Wang, Shaohe / Matsumoto, Kazue / Mehlferber, Madison M / Zhang, Guofeng / Aronova, Maria A / Yamada, Kenneth M

    Cells & development

    2023  Volume 177, Page(s) 203898

    Abstract: The basement membrane (BM) demarcating epithelial tissues undergoes rapid expansion to accommodate tissue growth and morphogenesis during embryonic development. To facilitate the secretion of bulky BM proteins, their mRNAs are polarized basally in the ... ...

    Abstract The basement membrane (BM) demarcating epithelial tissues undergoes rapid expansion to accommodate tissue growth and morphogenesis during embryonic development. To facilitate the secretion of bulky BM proteins, their mRNAs are polarized basally in the follicle epithelial cells of the Drosophila egg chamber to position their sites of production close to their deposition. In contrast, we observed the apical rather than basal polarization of all major BM mRNAs in the outer epithelial cells adjacent to the BM of mouse embryonic salivary glands using single-molecule RNA fluorescence in situ hybridization (smFISH). Moreover, electron microscopy and immunofluorescence revealed apical polarization of both the endoplasmic reticulum (ER) and Golgi apparatus, indicating that the site of BM component production was opposite to the site of deposition. At the apical side, BM mRNAs colocalized with ER, suggesting they may be co-translationally tethered. After microtubule inhibition, the BM mRNAs and ER became uniformly distributed rather than apically polarized, but they remained unchanged after inhibiting myosin II, ROCK, or F-actin, or after enzymatic disruption of the BM. Because Rab6 is generally required for Golgi-to-plasma membrane trafficking of BM components, we used lentivirus to express an mScarlet-tagged Rab6a in salivary gland epithelial cultures to visualize vesicle trafficking dynamics. We observed extensive bidirectional vesicle movements between Golgi at the apical side and the basal plasma membrane adjacent to the BM. Moreover, we showed that these vesicle movements depend on the microtubule motor kinesin-1 because very few vesicles remained motile after treatment with kinesore to compete for cargo-binding sites on kinesin-1. Overall, our work highlights the diverse strategies that different organisms use to secrete bulky matrix proteins: while Drosophila follicle epithelial cells strategically place their sites of BM protein production close to their deposition, mouse embryonic epithelial cells place their sites of production at the opposite end. Instead of spatial proximity, they use the microtubule cytoskeleton to mediate this organization as well as for the apical-to-basal transport of BM proteins.
    MeSH term(s) Animals ; Mice ; Basement Membrane/metabolism ; Kinesins/genetics ; Kinesins/metabolism ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; In Situ Hybridization, Fluorescence ; Microtubules/genetics ; Epithelial Cells/metabolism ; Drosophila/genetics ; Drosophila/metabolism ; Endoplasmic Reticulum/genetics ; Endoplasmic Reticulum/metabolism
    Chemical Substances Kinesins (EC 3.6.4.4) ; RNA, Messenger
    Language English
    Publishing date 2023-12-14
    Publishing country Netherlands
    Document type Journal Article ; Research Support, N.I.H., Intramural
    ISSN 2667-2901
    ISSN (online) 2667-2901
    DOI 10.1016/j.cdev.2023.203898
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  9. Article ; Online: Prognostic and immunological significance of peroxisome proliferator-activated receptor gamma in hepatocellular carcinoma based on multiple databases.

    Wang, Chaoban / Yu, Shiwen / Qian, Rengcheng / Chen, Shaohe / Dai, Chengjun / Shan, Xiaoou

    Translational cancer research

    2022  Volume 11, Issue 7, Page(s) 1938–1953

    Abstract: Background: Peroxisome proliferator-activated receptor gamma (PPARG) plays some roles in preventing liver disease progression to hepatocellular carcinoma. However, there is limited information about the function of PPARG of in hepatocellular carcinoma. ... ...

    Abstract Background: Peroxisome proliferator-activated receptor gamma (PPARG) plays some roles in preventing liver disease progression to hepatocellular carcinoma. However, there is limited information about the function of PPARG of in hepatocellular carcinoma. This study aimed to determine the significance of PPARG in immunological response and as a biomarker for hepatocellular carcinoma survival.
    Methods: We investigated the expression, prognosis, Kyoto Encyclopedia of Genes and Genomes/Gene Ontology biological process enrichment, and immune significance of PPARG using data from three databases-The Cancer Genome Atlas, International Cancer Genome Consortium, and Gene Expression Omnibus-through bioinformatics analysis as well as experimental verification in proliferation function of PPARG in HepG2 cell.
    Results: High PPARG expression in hepatocellular carcinoma tissues positively correlated with
    Conclusions: PPARG is upregulated in hepatocellular carcinoma and it correlates with a worse prognosis. Moreover, PPARG may play an important role in the cell proliferation, complement system and immune cell infiltration in hepatocellular carcinoma.
    Language English
    Publishing date 2022-10-07
    Publishing country China
    Document type Journal Article
    ZDB-ID 2901601-0
    ISSN 2219-6803 ; 2218-676X
    ISSN (online) 2219-6803
    ISSN 2218-676X
    DOI 10.21037/tcr-21-2853
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  10. Article ; Online: Localized Lysosome Exocytosis Helps Breach Tissue Barriers.

    Wang, Shaohe / Yamada, Kenneth M

    Developmental cell

    2017  Volume 43, Issue 4, Page(s) 377–378

    Abstract: Cell invasion across basement membrane barriers is important in both normal development and cancer metastasis. In this issue of Developmental Cell, Naegeli et al. (2017) identify a mechanism for breaching basement membranes. Localized lysosome exocytosis ...

    Abstract Cell invasion across basement membrane barriers is important in both normal development and cancer metastasis. In this issue of Developmental Cell, Naegeli et al. (2017) identify a mechanism for breaching basement membranes. Localized lysosome exocytosis fuels generation of large, invasive cellular protrusions that expand tiny basement membrane openings.
    Language English
    Publishing date 2017-11-20
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2054967-2
    ISSN 1878-1551 ; 1534-5807
    ISSN (online) 1878-1551
    ISSN 1534-5807
    DOI 10.1016/j.devcel.2017.11.005
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