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https://www.readbyqxmd.com/read/28448034/using-a-fluorescent-pcr-capillary-gel-electrophoresis-technique-to-genotype-crispr-cas9-mediated-knockout-mutants-in-a-high-throughput-format
#1
Muhammad Khairul Ramlee, Jing Wang, Alice M S Cheung, Shang Li
The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site...
April 8, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28447897/specific-hepatic-stellate-cell-penetrating-peptide-targeted-delivery-of-a-kla-peptide-reduces-collagen-accumulation-by-inducing-apoptosis
#2
Zhengrong Guo, Dong Li, Huanyan Peng, Jiwen Kang, Xiaoyu Jiang, Xiaoli Xie, Dianxing Sun, Huijing Jiang
Liver fibrosis is an aberrant wound-healing process to chronic hepatic inflammation and is characterized by excessive accumulation of extracellular matrix (ECM) that is produced by activated hepatic stellate cells (HSCs). Thus, activated HSCs play a key role in the pathogenesis of liver fibrosis and are a potential target for the treatment of liver fibrosis. Herein, we report that a specific HSC-penetrating peptide reduced collagen accumulation by inducing the apoptosis of HSC-T6 cells. We first screened HSC-specific transduction peptides and identified a novel HSC-targeted cell-penetrating peptide (HTP) that specifically interacted with HSC-T6 cells...
April 27, 2017: Journal of Drug Targeting
https://www.readbyqxmd.com/read/28447573/improving-neurorepair-in-stroke-brain-through-endogenous-neurogenesis-enhancing-drugs
#3
Kuo-Jen Wu, Seongjin Yu, Jea-Young Lee, Barry Hoffer, Yun Wang
Stroke induces not only cell death but also neurorepair. De novo neurogenesis has been found in the subventricular zone(SVZ) of adult mammalian brain days after stroke. Most of these newly generated cells die shortly after the insult. Recent studies have shown that pharmacological manipulation can improve the survival of endogenous neuroprogenitor cells and neural regeneration in stroke rats. As these drugs target the endogenous reparative processes which occur days after stroke, they may provide a prolonged window for stroke therapy...
April 26, 2017: Cell Transplantation
https://www.readbyqxmd.com/read/28446743/mammalian-target-of-rapamycin-mtor-regulates-transforming-growth-factor-%C3%AE-1-tgf-%C3%AE-1-induced-epithelial-mesenchymal-transition-via-decreased-pyruvate-kinase-m2-pkm2-expression-in-cervical-cancer-cells
#4
Ke-Yan Cheng, Min Hao
BACKGROUND Epithelial-mesenchymal transition (EMT) plays an important role in cancer tumorigenesis. Transforming growth factor β1 (TGF-β1) can induced EMT, which could increase tumor migration and invasion. Moreover, recent studies have been proven that mammalian target of rapamycin (mTOR) is a critical regulator of EMT. We investigated the mechanisms of mTOR in transforming growth factor β1 (TGF-β1)-induced EMT in cervical cancer cells. MATERIAL AND METHODS HeLa and SiHa cells were treated with 10 ng/ml TGF-β1 to induce EMT...
April 27, 2017: Medical Science Monitor: International Medical Journal of Experimental and Clinical Research
https://www.readbyqxmd.com/read/28445602/identification-of-a-novel-mirna-that-increases-transient-protein-expression-in-combination-with-valproic-acid
#5
Hermann-Josef Meyer, Dorothea Reilly, Scott E Martin, Athena W Wong
Transient gene expression in mammalian cells is an efficient process to produce recombinant proteins for various research applications and large molecule therapeutics development. For the first time, we report a screen to identify human microRNAs (miRNAs) that increase titers after polyethylenimine (PEI) mediated transient transfection of a HEK293 cell line. From a library of 875 miRNAs, we identified 2 miRNAs, miR-26a-5p and miR-337-5p, that increased human IgG1 (huIgG1) yields by 50% and 25%, respectively...
April 26, 2017: Biotechnology Progress
https://www.readbyqxmd.com/read/28442548/keratin-8-18-regulation-of-insulin-receptor-signaling-and-trafficking-in-hepatocytes-through-a-concerted-phosphoinositide-dependent-akt-and-rab5-modulation
#6
Alexandra Roux, Anne Loranger, Josée N Lavoie, Normand Marceau
Keratins (Ks) are epithelial cell intermediate filament (IF) proteins that are expressed as pairs in a differentiation-regulated manner. Hepatocyte IFs are made only of K8/K18 pairs, which means that a K8 loss in K8-null mice leads to degradation of K18. Functionally, there is accumulating evidence that IFs contribute to signaling platforms. Here, we investigate the role of K8/K18 IFs in the regulation of insulin receptor (IR) signaling and trafficking in hepatocytes. We find that the IR substrate 1 (IRS1)/PI3K/Akt signaling cascade-downstream of IR-display prolonged activation in K8-null compared with wild-type hepatocytes...
April 25, 2017: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/28442387/establishment-of-microrna-delivery-system-by-pp7-bacteriophage-like-particles-carrying-cell-penetrating-peptide
#7
Yanli Sun, Yanhua Sun, Ronglan Zhao
MicroRNAs have great therapeutic potential in cancer and other diseases. However, their instability and low in vivo delivery efficiency limits their application. Recombinant PP7 bacteriophage-based virus-like particles (VLPs) could protect microRNAs against rapid degradation by RNase by packaging specific exogenous pre-microRNAs using the pac site. Insertion of a cell-penetrating peptide (CPP) into the AB-loop of VLPs could significantly improve the delivery efficiency of microRNAs into mammalian cells. Unlike other microRNA delivery methods (viral or non-viral vectors), recombinant PP7 VLPs carrying a CPP and microRNA could be efficiently expressed in Escherichia coli using the one-plasmid double expression system...
April 22, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28440919/rev7-the-regulatory-subunit-of-pol%C3%AE-undergoes-uv-induced-and-cul4-dependent-degradation
#8
Audesh Bhat, Zhoushuai Qin, Guifen Wang, Wangyang Chen, Wei Xiao
In eukaryotic cells, Rev7 interacts with Rev3 and functions as a regulatory subunit of Polζ, a translesion DNA synthesis (TLS) polymerase. In addition to its role in TLS, mammalian Rev7, also known as Mad2B/Mad2L2, participates in multiple cellular activities including cell cycle progression and double-strand break repair through its interaction with several proteins. Here we show that in mammalian cells Rev7 undergoes ubiquitin/proteasome-mediated degradation upon UV irradiation in a time-dependent manner...
April 25, 2017: FEBS Journal
https://www.readbyqxmd.com/read/28439825/small-rna-profiling-by-next-generation-sequencing-using-high-definition-adapters
#9
Martina Billmeier, Ping Xu
Small RNAs (sRNAs) as key regulators of gene expression play fundamental roles in many biological processes. Next-generation sequencing (NGS) has become an important tool for sRNA discovery and profiling. However, NGS data often show bias for or against certain sequences which is mainly caused by adapter oligonucleotides that are ligated to sRNAs more or less efficiently by RNA ligases. In order to reduce ligation bias, High-definition (HD) adapters for the Illumina sequencing platform were developed. However, a large amount of direct 5' and 3' adapter ligation products are often produced when the current commercially available kits are used for cloning with HD adapters...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28438634/structural-insights-into-baf47-and-baf155-complex-formation
#10
Li Yan, Si Xie, Yongming Du, Chengmin Qian
Mammalian BAF complexes are a subfamily of SWI/SNF ATP-dependent chromatin remodelers that dynamically modulate chromatin structure to regulate fundamental cellular processes including gene transcription, cell cycle control and DNA damage response. So far many distinct BAF complexes have been identified with polymorphic assemblies of up to 15 subunits from 29 genes. The evolutionarily conserved BRG1/BRM, BAF47 and BAF155/BAF170 form a stable complex that carries out essential chromatin remodeling activity and therefore have been regarded as the core components of BAF complex...
April 21, 2017: Journal of Molecular Biology
https://www.readbyqxmd.com/read/28438183/semaphorin-plexin-signaling-influences-early-ventral-telencephalic-development-and-thalamocortical-axon-guidance
#11
Manuela D Mitsogiannis, Graham E Little, Kevin J Mitchell
BACKGROUND: Sensory processing relies on projections from the thalamus to the neocortex being established during development. Information from different sensory modalities reaching the thalamus is segregated into specialized nuclei, whose neurons then send inputs to cognate cortical areas through topographically defined axonal connections. Developing thalamocortical axons (TCAs) normally approach the cortex by extending through the subpallium; here, axonal navigation is aided by distributed guidance cues and discrete cell populations, such as the corridor neurons and the internal capsule (IC) guidepost cells...
April 24, 2017: Neural Development
https://www.readbyqxmd.com/read/28436127/tip110-deletion-impaired-embryonic-and-stem-cell-development-involving-down-regulation-of-stem-cell-factors-nanog-oct4-and-sox2
#12
Amanda Whitmill, Ying Liu, Khalid Amine Timani, Yinghua Niu, Johnny J He
HIV-1 Tat-interacting protein of 110 kDa, Tip110, plays important roles in multiple biological processes. In this study, we aimed to characterize the function of Tip110 in embryonic development. Transgenic mice lacking expression of a functional Tip110 gene (Tip110(-/-) ) died post-implantation, and Tip110(-/-) embryos exhibited developmental arrest between 8.5 and 9.5 days post coitum. However, in vitro cultures of Tip110(-/-) embryos showed that Tip110 loss did not impair embryo growth from the zygote to the blastocyst...
April 24, 2017: Stem Cells
https://www.readbyqxmd.com/read/28433890/il-37-induces-autophagy-in-hepatocellular-carcinoma-cells-by-inhibiting-the-pi3k-akt-mtor-pathway
#13
Ting-Ting Li, Di Zhu, Tong Mou, Zhen Guo, Jun-Liang Pu, Qing-Song Chen, Xu-Fu Wei, Zhong-Jun Wu
Autophagy is an intracellular "self-eating" process that is closely related to inflammation and cellular immunity. New studies indicate that autophagy is also involved in tumor suppression. The anti-inflammatory cytokine interleukin-37 (IL-37) has been shown to have tumor-suppressive abilities in hepatocellular carcinoma (HCC). Notably, autophagy appears to play a dual role in the development of HCC and may be involved in both tumorigenesis and tumor suppression. However, the potential role of IL-37 in autophagy is currently unknown...
April 20, 2017: Molecular Immunology
https://www.readbyqxmd.com/read/28432198/folate-dependent-methylation-of-septins-governs-ciliogenesis-during-neural-tube-closure
#14
Manami Toriyama, Michinori Toriyama, John B Wallingford, Richard H Finnell
Periconception maternal folic acid (vitamin B9) supplementation can reduce the prevalence of neural tube defects (NTDs), although just how folates benefit the developing embryo and promote closing of the neural tube and other morphologic processes during development remains unknown. Folate contributes to a 1-carbon metabolism, which is essential for purine biosynthesis and methionine recycling and affects methylation of DNA, histones, and nonhistone proteins. Herein, we used animal models and cultured mammalian cells to demonstrate that disruption of the methylation pathway mediated by folate compromises normal neural tube closure (NTC) and ciliogenesis...
April 21, 2017: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/28431403/association-of-the-vdac3-gene-polymorphism-with-sperm-count-in-han-chinese-population-with-idiopathic-male-infertility
#15
Lianjun Pan, Qingzhen Liu, Jingyun Li, Wei Wu, Xinru Wang, Dan Zhao, Jiehua Ma
Voltage-dependent anion channel (VDAC) is a multifunctional channel protein across the outer mitochondrial membrane of somatic cells and participates in many physiological and pathophysiological processes. Up to now, only a few studies, including our previous studies, showed that VDAC exists in mammalian spermatozoa and is involved in spermatogenesis and sperm functions. There is no report about VDAC genetic variants in germinal tissues or cells. To investigate the possible association between VDAC genetic variants and human sperm quality, we performed semen analysis and variant Genotyping of VDAC3 subtype (rs7004637, rs16891278 and rs6773) of 523 Han-Chinese males with idiopathic infertility respectively by computer assisted semen analysis (CASA) and single nucleotide polymorphism (SNP) Genotyping assay...
April 6, 2017: Oncotarget
https://www.readbyqxmd.com/read/28431041/bidirectional-approaches-for-optogenetic-regulation-of-gene-expression-in-mammalian-cells-using-arabidopsis-cryptochrome-2
#16
Gopal P Pathak, Jessica I Spiltoir, Camilla Höglund, Lauren R Polstein, Sari Heine-Koskinen, Charles A Gersbach, Jari Rossi, Chandra L Tucker
Optogenetic tools allow regulation of cellular processes with light, which can be delivered with spatiotemporal resolution. In previous work, we used cryptochrome 2 (CRY2) and CIB1, Arabidopsis proteins that interact upon light illumination, to regulate transcription with light in yeast. While adopting this approach to regulate transcription in mammalian cells, we observed light-dependent redistribution and clearing of CRY2-tethered proteins within the nucleus. The nuclear clearing phenotype was dependent on the presence of a dimerization domain contained within the CRY2-fused transcriptional activators...
April 20, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28430595/minus-end-directed-kinesin-14-kifc1-regulates-the-positioning-and-architecture-of-the-golgi-apparatus
#17
Zhen-Yu She, Meng-Ying Pan, Fu-Qing Tan, Wan-Xi Yang
The Golgi apparatus is the central organelle along the eukaryotic secretory and endocytic pathway. In non-polarized mammalian cells, the Golgi complex is usually located proximal to the nucleus at the cell center and is closely associated with the microtubule organizing center. Microtubule networks are essential in the organization and central localization of the Golgi apparatus, but the molecular basis underlying these processes are poorly understood. Here we reveal that minus end-directed kinesin-14 KIFC1 proteins are required for the structural integrity and positioning of the Golgi complex in non-polarized mammalian cells...
April 5, 2017: Oncotarget
https://www.readbyqxmd.com/read/28430160/subverting-host-cell-p21-activated-kinase-a-case-of-convergent-evolution-across-pathogens
#18
REVIEW
Simona John Von Freyend, Terry Kwok-Schuelein, Hans Netter, Gholamreza Haqshenas, Jean-Philippe Semblat, Christian Doerig
Intracellular pathogens have evolved a wide range of strategies to not only escape from the immune systems of their hosts, but also to directly exploit a variety of host factors to facilitate the infection process. One such strategy is to subvert host cell signalling pathways to the advantage of the pathogen. Recent research has highlighted that the human serine/threonine kinase PAK, or p21-activated kinase, is a central component of host-pathogen interactions in many infection systems involving viruses, bacteria, and eukaryotic pathogens...
April 21, 2017: Pathogens
https://www.readbyqxmd.com/read/28429463/global-mapping-of-protein-lipid-interactions-by-using-modified-choline-containing-phospholipids-metabolically-synthesized-in-live-cells
#19
Danyang Wang, Shubo Du, Amaury Cazenave-Gassiot, Jingyan Ge, Jun-Seok Lee, Markus R Wenk, Shao Q Yao
The protein-lipid interaction is an essential metabolic process that mediates cellular signaling and functions. Existing strategies for large-scale mapping studies of the protein-lipid interaction fall short in their incompatibility with metabolic incorporation or inability to remove unwanted interferences from lipidated proteins. By incorporating an alkyne-containing choline head group and a diazirine-modified fatty acid simultaneously into choline-containing phospholipids synthesized from live mammalian cells, protein-phospholipid interactions have been successfully imaged in live cells...
April 21, 2017: Angewandte Chemie
https://www.readbyqxmd.com/read/28428954/uncovering-the-roles-of-septins-in-cilia
#20
REVIEW
Oliva Palander, Maha El-Zeiry, William S Trimble
Septins are a family of GTP-binding proteins that associate with cellular membranes and the cytoskeleton. Their ability to polymerize into filamentous structures permits them to serve as diffusion barriers for membrane proteins and as multi-molecular scaffolds that recruit components of signaling pathways. At the cellular level, septins contribute to the regulation of numerous processes, including cytokinesis, cell polarity, cell migration, and many others. In this review, we discuss emerging evidence for roles of mammalian septins in the biogenesis and function of flagella and cilia, and how this may impact human diseases such as ciliopathies...
2017: Frontiers in Cell and Developmental Biology
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