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https://www.readbyqxmd.com/read/28219395/efficient-precise-knockin-with-a-double-cut-hdr-donor-after-crispr-cas9-mediated-double-stranded-dna-cleavage
#1
Jian-Ping Zhang, Xiao-Lan Li, Guo-Hua Li, Wanqiu Chen, Cameron Arakaki, Gary D Botimer, David Baylink, Lu Zhang, Wei Wen, Ya-Wen Fu, Jing Xu, Noah Chun, Weiping Yuan, Tao Cheng, Xiao-Bing Zhang
BACKGROUND: Precise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. However, HDR efficiency remains low in some cell types, including some of great research and clinical interest, such as human induced pluripotent stem cells (iPSCs). RESULTS: Here, we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs...
February 20, 2017: Genome Biology
https://www.readbyqxmd.com/read/28202041/the-microtubule-plus-end-tracking-protein-tacc3-promotes-persistent-axon-outgrowth-and-mediates-responses-to-axon-guidance-signals-during-development
#2
Burcu Erdogan, Garrett M Cammarata, Eric J Lee, Benjamin C Pratt, Andrew F Francl, Erin L Rutherford, Laura Anne Lowery
BACKGROUND: Formation of precise neuronal connections requires proper axon guidance. Microtubules (MTs) of the growth cone provide a critical driving force during navigation of the growing ends of axons. Pioneer MTs and their plus-end tracking proteins (+TIPs) are thought to play integrative roles during this navigation. TACC3 is a + TIP that we have previously implicated in regulating MT dynamics within axons. However, the role of TACC3 in axon guidance has not been previously explored...
February 15, 2017: Neural Development
https://www.readbyqxmd.com/read/28196706/the-role-of-cell-cycle-progression-for-the-apoptosis-of-cancer-cells-induced-by-palladium-ii-saccharinate-complexes-of-terpyridine
#3
Omer Kacar, Buse Cevatemre, Ibrahim Hatipoglu, Nazli Arda, Engin Ulukaya, Veysel T Yilmaz, Ceyda Acilan
OBJECTIVES: Palladium complexes are potent and less toxic molecules in comparison to other metal based agents. Here, we characterized two palladium(II) saccharinate complexes with terpyridine for their cell cycle specificity. MATERIALS AND METHODS: Cells were arrested at G1, G1/S boundary or mitosis using mimosine, double-Thymidine block, aphidicolin, nocodazole or colcemid, and evaluated based on morphology and flow cytometry. Synchronized cells were treated with the Pd(II) complexes, and viability was measured via MTT assay...
January 31, 2017: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/28179028/betanodavirus-like-particles-enter-host-cells-via-clathrin-mediated-endocytosis-in-a-cholesterol-ph-and-cytoskeleton-dependent-manner
#4
Runqing Huang, Guohua Zhu, Jing Zhang, Yuxiong Lai, Yu Xu, Jianguo He, Junfeng Xie
Betanodavirus, also referred to nervous necrosis virus (NNV), is the causative agent of the fatal disease, viral nervous necrosis and has brought significant economic losses in marine and freshwater cultured fish, especially larvae and juveniles. Here, we used an established invasion model with virus-like particle (VLP)-cells, mimicking orange-spotted grouper nervous necrosis virus (OGNNV), to investigate the crucial events of virus entry. VLP were observed in the perinuclear regions of Asian sea bass (SB) cells within 1...
February 8, 2017: Veterinary Research
https://www.readbyqxmd.com/read/28177917/cytoskeleton-associated-protein-5-and-clathrin-heavy-chain-binding-regulates-spindle-assembly-in-mouse-oocytes
#5
Angeleem Lu, Cheng-Jie Zhou, Dong-Hui Wang, Zhe Han, Xiang-Wei Kong, Yu-Zhen Ma, Zhi-Zhong Yun, Cheng-Guang Liang
Mammalian oocyte meiotic maturation is the precondition of early embryo development. Lots of microtubules (MT)-associated proteins participate in oocyte maturation process. Cytoskeleton-associated protein 5 (CKAP5) is a member of the XMAP215 family that regulates microtubule dynamics during mitosis. However, its role in meiosis has not been fully studied. Here, we investigated the function of CKAP5 in mouse oocyte meiotic maturation and early embryo development. Western blot showed that CKAP5 expression increased from GVBD, maintaining at high level at metaphase, and decreased after late 1-cell stage...
February 4, 2017: Oncotarget
https://www.readbyqxmd.com/read/28174522/in-vivo-imaging-of-mitochondrial-transport-in-single-axon-regeneration-of-zebrafish-mauthner-cells
#6
Yang Xu, Min Chen, Bingbing Hu, Rongchen Huang, Bing Hu
Mitochondrial transport is essential for neuronal function, but the evidence of connections between mitochondrial transport and axon regeneration in the central nervous system (CNS) of living vertebrates remains limited. Here, we developed a novel model to explore mitochondrial transport in a single Mauthner axon (M axon) of zebrafish with non-invasive in vivo imaging. To confirm the feasibility of using this model, we treated labeled zebrafish with nocodazole and demonstrated that it could disrupt mitochondrial transport...
2017: Frontiers in Cellular Neuroscience
https://www.readbyqxmd.com/read/28118058/smc1%C3%AE-is-required-for-activation-of-sac-during-mouse-oocyte-meiosis
#7
Yilong Miao, Changyin Zhou, Zhaokang Cui, Xiaoxin Dai, Mianqun Zhang, Yajuan Lu, Bo Xiong
Smc1β is a meiosis-specific cohesin subunit that is essential for sister chromatid cohesion and DNA recombination. Previous studies have shown that Smc1β-deficient mice in both sexes are sterile. Ablation of Smc1β during male meiosis leads to the blockage of spermatogenesis in pachytene stage, and ablation of Smc1β during female meiosis generates a highly error-prone oocyte although it could develop to metaphase II stage. However, the underlying mechanisms regarding how Smc1β maintains the correct meiotic progression in mouse oocytes have not been clearly defined...
January 24, 2017: Cell Cycle
https://www.readbyqxmd.com/read/28112196/the-kinetochore-dependent-and-independent-formation-of-the-cdc20-mad2-complex-and-its-functions-in-hela-cells
#8
Jianquan Li, Nanmao Dang, Daniel James Wood, Jun-Yong Huang
The mitotic checkpoint complex (MCC) is formed from two sub-complexes of CDC20-MAD2 and BUBR1-BUB3, and current models suggest that it is generated exclusively by the kinetochores after nuclear envelope breakdown (NEBD). However, neither sub-complex has been visualised in vivo, and when and where they are formed during the cell cycle and their response to different SAC conditions remains elusive. Using single cell analysis in HeLa cells, we show that the CDC20-MAD2 complex is cell cycle regulated with a "Bell" shaped profile and peaks at prometaphase...
January 23, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28049774/synchronization-and-arrest-of-the-budding-yeast-cell-cycle-using-chemical-and-genetic-methods
#9
Adam P Rosebrock
The cell cycle of budding yeast can be arrested at specific positions by different genetic and chemical methods. These arrests enable study of cell cycle phase-specific phenotypes that would be missed during examination of asynchronous cultures. Some methods for arrest are reversible, with kinetics that enable release of cells back into a synchronous cycling state. Benefits of chemical and genetic methods include scalability across a large range of culture sizes from a few milliliters to many liters, ease of execution, the absence of specific equipment requirements, and synchronization and release of the entire culture...
January 3, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28017899/nocodazole-treatment-interrupted-brucella-abortus-invasion-in-raw-264-7-cells-and-successfully-attenuated-splenic-proliferation-with-enhanced-inflammatory-response-in-mice
#10
Alisha Wehdnesday Bernardo Reyes, Huynh Tan Hop, Lauren Togonon Arayan, Tran Xuan Ngoc Huy, Wongi Min, Hu Jang Lee, Hong Hee Chang, Suk Kim
Brucellosis is one of the most important and widespread zoonosis worldwide responsible for serious economic losses and considerable public health burden. In this study, we investigated the modulatory effect of a microtubule-inhibitor, nocodazole, on B. abortus infection in murine macrophages and in a mouse model. Nocodazole activated macrophages and directly inhibited the growth of Brucella in a dose-dependent manner. Nocodazole increased adhesion but reduced invasion and intracellular growth of Brucella in macrophages although it did not affect co-localization of Brucella with LAMP-1...
December 23, 2016: Microbial Pathogenesis
https://www.readbyqxmd.com/read/28011649/newcastle-disease-virus-induces-stable-formation-of-bona-fide-stress-granules-to-facilitate-viral-replication-through-manipulating-host-protein-translation
#11
Yingjie Sun, Luna Dong, Shengqing Yu, Xiaoxu Wang, Hang Zheng, Pin Zhang, Chunchun Meng, Yuan Zhan, Lei Tan, Cuiping Song, Xusheng Qiu, Guijun Wang, Ying Liao, Chan Ding
Mammalian cells respond to various environmental stressors to form stress granules (SGs) by arresting cytoplasmic mRNA, protein translation element, and RNA binding proteins. Virus-induced SGs function in different ways, depending on the species of virus; however, the mechanism of SG regulation of virus replication is not well understood. In this study, Newcastle disease virus (NDV) triggered stable formation of bona fide SGs on HeLa cells through activating the protein kinase R (PKR)/eIF2α pathway. NDV-induced SGs contained classic SG markers T-cell internal antigen (TIA)-1, Ras GTPase-activating protein-binding protein (G3BP)-1, eukaryotic initiation factors, and small ribosomal subunit, which could be disassembled in the presence of cycloheximide...
December 23, 2016: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/28004475/structure-based-approaches-for-the-design-of-benzimidazole-2-carbamate-derivatives-as-tubulin-polymerization-inhibitors
#12
Rodrigo Aguayo-Ortiz, Lucia Cano-González, Rafael Castillo, Alicia Hernández-Campos, Laura Domínguez
Microtubules are highly dynamic assemblies of α/β-tubulin heterodimers whose polymerization inhibition is among one of the most successful approaches for anticancer drug development. Overexpression of the class I (βI) and class III (βIII) β-tubulin isotypes in breast and lung cancers and the highly expressed class VI (βVI) β-tubulin isotype in normal blood cells have increased the interest for designing specific tubulin-binding anticancer therapies. To this end, we employed our previously proposed model of the β-tubulin-nocodazole complex, supported by the recently determined X-ray structure, to identify the fundamental structural differences between β-tubulin isotypes...
December 22, 2016: Chemical Biology & Drug Design
https://www.readbyqxmd.com/read/28004015/influenza-infection-modulates-vesicular-trafficking-and-induces-golgi-complex-disruption
#13
Vibha Yadav, Antonito T Panganiban, Kerstin Honer Zu Bentrup, Thomas G Voss
Influenza A virus (IFV) replicates its genome in the nucleus of infected cells and uses the cellular protein transport system for genome trafficking from the nucleus to the plasma membrane. However, many details of the mechanism of this process, and its relationship to subsequent cytoplasmic virus trafficking, have not been elucidated. We examined the effect of nuclear transport inhibitors Leptomycin B (LB), 5,6 dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), the vesicular transport inhibitor Brefeldin A (BFA), the caspase inhibitor ZWEHD, and microtubule inhibitor Nocodazole (NOC) on virus replication and intracellular trafficking of viral nucleoprotein (NP) from the nucleus to the ER and Golgi...
December 2016: Virusdisease
https://www.readbyqxmd.com/read/27997747/dynamics-of-the-sealing-zone-in-cultured-osteoclasts
#14
Sarit Batsir, Benjamin Geiger, Zvi Kam
Bone resorption by osteoclasts (OCs) depends on the formation and stability of the sealing zone (SZ), a peripheral belt of actin and integrin-based podosomes. Recent studies demonstrated that the SZ is a highly dynamic structure, undergoing cycles of assembly and disassembly. In this study, we explored the mechanisms underlying the regulation of SZ stability and reorganization in OCs cultured on glass slides, and forming an SZ-like podosome belt (SZL). By monitoring this belt in cultured RAW264.7 cells expressing GFP-tagged actin, we show here that SZL stability is usually locally regulated, and its dissociation, occurring mostly in concave segments, is manifested in the loss of both podosome coherence, and actin belt continuity...
February 2017: Cytoskeleton
https://www.readbyqxmd.com/read/27994056/inducible-inhibition-of-g%C3%AE-%C3%AE-reveals-localization-dependent-functions-at-the-plasma-membrane-and-golgi
#15
Lauren M Klayman, Philip B Wedegaertner
Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on βγ signaling at the Golgi, where βγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used βγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system...
February 3, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/27991556/kif2a-regulates-the-spindle-assembly-and-the-metaphase-i-anaphase-i-transition-in-mouse-oocyte
#16
Ming-Huang Chen, Yu Liu, Ya-Long Wang, Rui Liu, Bai-Hui Xu, Fei Zhang, Fei-Ping Li, Lin Xu, Yan-Hong Lin, Shu-Wen He, Bao-Qiong Liao, Xian-Pei Fu, Xiao-Xue Wang, Xiang-Jun Yang, Hai-Long Wang
KIF2A, a member of the kinesin-13 family, has been reported to play a role in spindle assembly in mitosis. However, its function in mammalian meiosis remains unknown. In this research, we examined the expression, localization and function of KIF2A during mouse oocyte meiosis. KIF2A was expressed in some key stages in mouse oocyte meiosis. Immunofluorescent staining showed that KIF2A distributed in the germinal vesicle at the germinal vesicle stage and as the spindle assembling after meiosis resumption, KIF2A gradually accumulated to the entire spindle...
December 19, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27942359/cytolethal-distending-toxin-from-campylobacter-jejuni-requires-the-cytoskeleton-for-toxic-activity
#17
Estela T Méndez-Olvera, Jaime A Bustos-Martínez, Yolanda López-Vidal, Antonio Verdugo-Rodríguez, Daniel Martínez-Gómez
BACKGROUND: Campylobacter jejuni is one of the major causes of infectious diarrhea worldwide. The distending cytolethal toxin (CDT) of Campylobacter spp. interferes with normal cell cycle progression. This toxic effect is considered a result of DNase activity that produces chromosomal DNA damage. To perform this event, the toxin must be endocytosed and translocated to the nucleus. OBJECTIVES: The aim of this study was to evaluate the role of the cytoskeleton in the translocation of CDT to the nucleus...
October 2016: Jundishapur Journal of Microbiology
https://www.readbyqxmd.com/read/27941677/the-enrichment-of-survivin-in-exosomes-from-breast-cancer-cells-treated-with-paclitaxel-promotes-cell-survival-and-chemoresistance
#18
Bridget T Kreger, Eric R Johansen, Richard A Cerione, Marc A Antonyak
The generation and release of membrane-enclosed packets from cancer cells, called extracellular vesicles (EVs), play important roles in propagating transformed phenotypes, including promoting cell survival. EVs mediate their effects by transferring their contents, which include specific proteins and nucleic acids, to target cells. However, how the cargo and function of EVs change in response to different stimuli remains unclear. Here, we discovered that treating highly aggressive MDAMB231 breast cancer cells with paclitaxel (PTX), a chemotherapy that stabilizes microtubules, causes them to generate a specific class of EV, namely exosomes, that are highly enriched with the cell survival protein and cancer marker, Survivin...
December 9, 2016: Cancers
https://www.readbyqxmd.com/read/27885657/cdc25b-is-involved-in-the-centrosomal-microtubule-nucleation-in-two-cell-stage-mouse-embryos
#19
Cheng Cui, Tianxia Zang, Yu Cao, Xin Qin, Xuewei Zhang
CDC25B has been demonstrated to activate the complex of CDK1/Cyclin B and trigger mitosis. We have recently demonstrated that p-CDC25B-Ser351 is located at the centrosomes of mouse oocytes and contributes to the release of mouse oocytes from prophase I arrest. But much less is known about CDC25B function at the centrosome in two-cell stage mouse embryos. Here we investigate the effect of CDC25B regulating the microtubules nucleation. Microinjection of anti-CDC25B antibody caused aberrant microtubule nucleation...
December 2016: Development, Growth & Differentiation
https://www.readbyqxmd.com/read/27875556/mxa-is-a-novel-regulator-of-endosome-associated-transcriptional-signaling-by-bone-morphogenetic-proteins-4-and-9-bmp4-and-bmp9
#20
Huijuan Yuan, Pravin B Sehgal
There is confusion about the role that IFN-α plays in the pathogenesis of pulmonary arterial hypertension (PAH) with different investigators reporting a causative or a protective role. There is now clear evidence in PAH pathogenesis for the involvement of BMP4 and BMP9 signaling, and its disruption by mutations in BMPR2. In the present study, we investigated MxA, an IFN-α-inducible cytoplasmic dynamin-family GTPase for effects on BMP4/9 signaling, including in the presence of PAH-disease-associated mutants of BMPR2...
2016: PloS One
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