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https://www.readbyqxmd.com/read/28212749/comparative-analysis-of-single-cell-rna-sequencing-methods
#1
Christoph Ziegenhain, Beate Vieth, Swati Parekh, Björn Reinius, Amy Guillaumet-Adkins, Martha Smets, Heinrich Leonhardt, Holger Heyn, Ines Hellmann, Wolfgang Enard
Single-cell RNA sequencing (scRNA-seq) offers new possibilities to address biological and medical questions. However, systematic comparisons of the performance of diverse scRNA-seq protocols are lacking. We generated data from 583 mouse embryonic stem cells to evaluate six prominent scRNA-seq methods: CEL-seq2, Drop-seq, MARS-seq, SCRB-seq, Smart-seq, and Smart-seq2. While Smart-seq2 detected the most genes per cell and across cells, CEL-seq2, Drop-seq, MARS-seq, and SCRB-seq quantified mRNA levels with less amplification noise due to the use of unique molecular identifiers (UMIs)...
February 16, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28211892/high-throughput-in-situ-cell-electroporation-microsystem-for-parallel-delivery-of-single-guide-rnas-into-mammalian-cells
#2
Shengtai Bian, Yicen Zhou, Yawei Hu, Jing Cheng, Xiaofang Chen, Youchun Xu, Peng Liu
Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells...
February 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28211871/expression-level-is-a-key-determinant-of-e2f1-mediated-cell-fate
#3
Igor Shats, Michael Deng, Adam Davidovich, Carolyn Zhang, Jungeun S Kwon, Dinesh Manandhar, Raluca Gordân, Guang Yao, Lingchong You
The Rb/E2F network has a critical role in regulating cell cycle progression and cell fate decisions. It is dysfunctional in virtually all human cancers, because of genetic lesions that cause overexpression of activators, inactivation of repressors, or both. Paradoxically, the downstream target of this network, E2F1, is rarely strongly overexpressed in cancer. E2F1 can induce both proliferation and apoptosis but the factors governing these critical cell fate decisions remain unclear. Previous studies have focused on qualitative mechanisms such as differential cofactors, posttranslational modification or state of other signaling pathways as modifiers of the cell fate decisions downstream of E2F1 activation...
February 17, 2017: Cell Death and Differentiation
https://www.readbyqxmd.com/read/28211524/epigenetic-and-genetic-dissections-of-uv-induced-global-gene-dysregulation-in-skin-cells-through-multi-omics-analyses
#4
Yao Shen, Milda Stanislauskas, Gen Li, Deyou Zheng, Liang Liu
To elucidate the complex molecular mechanisms underlying the adverse effects UV radiation (UVR) on skin homeostasis, we performed multi-omics studies to characterize UV-induced genetic and epigenetic changes. Human keratinocytes from a single donor treated with or without UVR were analyzed by RNA-seq, exome-seq, and H3K27ac ChIP-seq at 4 h and 72 h following UVR. Compared to the relatively moderate mutagenic effects of UVR, acute UV exposure induced substantial epigenomic and transcriptomic alterations, illuminating a previously underappreciated role of epigenomic and transcriptomic instability in skin pathogenesis...
February 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28211111/regulation-of-nrf-operon-expression-in-pathogenic-enteric-bacteria-sequence-divergence-reveals-new-regulatory-complexity
#5
Rita E Godfrey, David J Lee, Stephen J W Busby, Douglas F Browning
The Escherichia coli K-12 nrf operon encodes a periplasmic nitrite reductase, the expression of which is driven from a single promoter, pnrf. Expression from pnrf is activated by the FNR transcription factor in response to anaerobiosis and further increased in response to nitrite by the response regulator proteins, NarL and NarP. FNR-dependent transcription is suppressed by the binding of two nucleoid associated proteins, IHF and Fis. As Fis levels increase in cells grown in rich medium, the positioning of its binding site, overlapping the promoter -10 element, ensures that pnrf is sharply repressed...
February 17, 2017: Molecular Microbiology
https://www.readbyqxmd.com/read/28207326/micro-rna-146a-but-not-irak1-is-associated-with-rheumatoid-arthritis-in-the-tunisian-population
#6
Hana Ben Hassine, Asma Boumiza, Rim Sghiri, Khadija Baccouche, Imen Boussaid, Ahlem Atig, Zahid Shakoor, Elyes Bouajina, Ramzi Zemni
BACKGROUND: Rheumatoid arthritis (RA) is characterized by the production of an array of proinflammatory cytokines through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. Interleukin-1 receptor (IL-1R) and Toll-like receptors contain a common cytoplasmic motif the Toll/IL-1R (TIR) homology domain. This motif is required for NF-κB activation. IL-1R-associated kinase 1 (IRAK1) is a key adapter molecule recruited during the signaling cascade of the TIR...
February 2017: Genetic Testing and Molecular Biomarkers
https://www.readbyqxmd.com/read/28207000/spatial-transcriptomic-analysis-of-cryosectioned-tissue-samples-with-geo-seq
#7
Jun Chen, Shengbao Suo, Patrick Pl Tam, Jing-Dong J Han, Guangdun Peng, Naihe Jing
Conventional gene expression studies analyze multiple cells simultaneously or single cells, for which the exact in vivo or in situ position is unknown. Although cellular heterogeneity can be discerned when analyzing single cells, any spatially defined attributes that underpin the heterogeneous nature of the cells cannot be identified. Here, we describe how to use Geo-seq, a method that combines laser capture microdissection (LCM) and single-cell RNA-seq technology. The combination of these two methods enables the elucidation of cellular heterogeneity and spatial variance simultaneously...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28205560/alkb-homolog-3-mediated-trna-demethylation-promotes-protein-synthesis-in-cancer-cells
#8
Yuko Ueda, Ikumi Ooshio, Yasuyuki Fusamae, Kaori Kitae, Megumi Kawaguchi, Kentaro Jingushi, Hiroaki Hase, Kazuo Harada, Kazumasa Hirata, Kazutake Tsujikawa
The mammalian AlkB homolog (ALKBH) family of proteins possess a 2-oxoglutarate- and Fe(II)-dependent oxygenase domain. A similar domain in the Escherichia coli AlkB protein catalyzes the oxidative demethylation of 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) in both DNA and RNA. AlkB homolog 3 (ALKBH3) was also shown to demethylate 1-meA and 3-meC (induced in single-stranded DNA and RNA by a methylating agent) to reverse the methylation damage and retain the integrity of the DNA/RNA. We previously reported the high expression of ALKBH3 in clinical tumor specimens and its involvement in tumor progression...
February 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28205168/crispr-cas9-mediated-generation-of-niemann-pick-c1-knockout-cell-line
#9
Ximing Du, Ivan Lukmantara, Hongyuan Yang
Generating a cholesterol storage phenotype of Niemann-Pick Type C (NPC) disease is important for investigating the mechanisms of intracellular cholesterol trafficking, as well as screening drugs for potential treatment of NPC disease. The use of the CRISPR/Cas9 technology to knockout specific genes within the genome of mammals has become routine in the past few years. Here, we describe a protocol for producing a cellular NPC cholesterol storage phenotype in HeLa cells using the CRISPR-Cas9 system to disrupt the NPC1 gene...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28203683/identification-of-unique-venous-thromboembolism-susceptibility-variants-in-african-americans
#10
John A Heit, Sebastian M Armasu, Bryan M McCauley, Iftikhar J Kullo, Hugues Sicotte, Jyotishman Pathak, Christopher G Chute, Omri Gottesman, Erwin P Bottinger, Joshua C Denny, Dan M Roden, Rongling Li, Marylyn D Ritchie, Mariza de Andrade
To identify novel single nucleotide polymorphisms (SNPs) associated with venous thromboembolism (VTE) in African-Americans (AAs), we performed a genome-wide association study (GWAS) of VTE in AAs using the Electronic Medical Records and Genomics (eMERGE) Network, comprised of seven sites each with DNA biobanks (total ~39,200 unique DNA samples) with genome-wide SNP data (imputed to 1000 Genomes Project cosmopolitan reference panel) and linked to electronic health records (EHRs). Using a validated EHR-driven phenotype extraction algorithm, we identified VTE cases and controls and tested for an association between each SNP and VTE using unconditional logistic regression, adjusted for age, sex, stroke, site-platform combination and sickle cell risk genotype...
February 16, 2017: Thrombosis and Haemostasis
https://www.readbyqxmd.com/read/28203566/increased-spontaneous-recombination-in-rnase-h2-deficient-cells-arises-from-multiple-contiguous-rnmps-and-not-from-single-rnmp-residues-incorporated-by-dna-polymerase-epsilon
#11
Anastasiya Epshtein, Catherine J Potenski, Hannah L Klein
Ribonucleotides can become embedded in DNA from insertion by DNA polymerases, failure to remove Okazaki fragment primers, R-loops that can prime replication, and RNA/cDNA-mediated recombination. RNA:DNA hybrids are removed by RNase H enzymes. Single rNMPs in DNA are removed by RNase H2 and if they remain on the leading strand, can lead to mutagenesis in a Top1-dependent pathway. rNMPs in DNA can also stimulate genome instability, among which are homologous recombination gene conversion events. We previously found that, similar to the rNMP-stimulated mutagenesis, rNMP-stimulated recombination was also Top1-dependent...
June 2016: Microbial Cell
https://www.readbyqxmd.com/read/28203244/pistil-transcriptome-analysis-to-disclose-genes-and-gene-products-related-to-aposporous-apomixis-in-hypericum-perforatum-l
#12
Giulio Galla, Sara Zenoni, Linda Avesani, Lothar Altschmied, Paride Rizzo, Timothy F Sharbel, Gianni Barcaccia
Unlike sexual reproduction, apomixis encompasses a number of reproductive strategies, which permit maternal genome inheritance without genetic recombination and syngamy. The key biological features of apomixis are the circumvention of meiosis (i.e., apomeiosis), the differentiation of unreduced embryo sacs and egg cells, and their autonomous development in functional embryos through parthenogenesis, and the formation of viable endosperm either via fertilization-independent means or following fertilization with a sperm cell...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28202516/modeling-the-genetic-regulation-of-cancer-metabolism-interplay-between-glycolysis-and-oxidative-phosphorylation
#13
Linglin Yu, Mingyang Lu, Dongya Jia, Jianpeng Ma, Eshel Ben-Jacob, Herbert Levine, Benny A Kaipparettu, Jose' N Onuchic
Abnormal metabolism is a hallmark of cancer, yet its regulation remains poorly understood. Cancer cells were considered to utilize primarily glycolysis for ATP production, referred to as the Warburg effect. However, recent evidence suggests that oxidative phosphorylation (OXPHOS) plays a crucial role during cancer progression. Here we utilized a systems biology approach to decipher the regulatory principle of glycolysis and OXPHOS. Integrating information from literature, we constructed a regulatory network of genes and metabolites from which we extracted a core circuit containing HIF-1, AMPK, and ROS...
February 15, 2017: Cancer Research
https://www.readbyqxmd.com/read/28202040/subcellular-localization-and-interactions-of-infectious-salmon-anemia-virus-isav-m1-and-nep-as-well-as-host-hsc70
#14
Wenting Zhang, Chengzhi Cai, Li Lin, Yizhi Jane Tao, Meilin Jin
BACKGROUND: Infectious salmon anemia virus (ISAV) is an important fish pathogen that causes high mortality in farmed Atlantic salmon. The ISAV genome consists of eight single-stranded, negative-sense RNA segments. The six largest segments contain one open reading frame (ORF) each, and encode three polymerase proteins, nucleoprotein, fusion protein, and hemagglutinin esterase protein. The two smallest segments contain more than one ORF each. The segment 7 encodes non-structural protein 1 (NS1) and nuclear export protein (NEP), while segment 8 encodes matrix protein 1 and 2 (M1 and M2)...
February 15, 2017: Virology Journal
https://www.readbyqxmd.com/read/28202026/an-indirect-enzyme-linked-immunosorbent-assay-for-the-identification-of-antibodies-to-senecavirus-a-in-swine
#15
Cheryl M T Dvorak, Zeynep Akkutay-Yoldar, Suzanne R Stone, Steven J P Tousignant, Fabio A Vannucci, Michael P Murtaugh
BACKGROUND: Senecavirus A (SVA), a member of the family Picornaviridae, genus Senecavirus, is a recently identified single-stranded RNA virus closely related to members of the Cardiovirus genus. SVA was originally identified as a cell culture contaminant and was not associated with disease until 2007 when it was first observed in pigs with Idiopathic Vesicular Disease (IVD). Vesicular disease is sporadically observed in swine, is not debilitating, but is significant due to its resemblance to foreign animal diseases, such as foot-and-mouth disease (FMD), whose presence would be economically devastating to the United States...
February 15, 2017: BMC Veterinary Research
https://www.readbyqxmd.com/read/28198371/method-for-dual-viral-vector-mediated-crispr-cas9-gene-disruption-in-primary-human-endothelial-cells
#16
Haixia Gong, Menglin Liu, Jeff Klomp, Bradley J Merrill, Jalees Rehman, Asrar B Malik
Human endothelial cells (ECs) are widely used to study mechanisms of angiogenesis, inflammation, and endothelial permeability. Targeted gene disruption induced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated Protein 9 (Cas9) nuclease gene editing is potentially an important tool for definitively establishing the functional roles of individual genes in ECs. We showed that co-delivery of adenovirus encoding EGFP-tagged Cas9 and lentivirus encoding a single guide RNA (sgRNA) in primary human lung microvascular ECs (HLMVECs) disrupted the expression of the Tie2 gene and protein...
February 15, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28198357/cgcorrect-a-method-to-correct-for-confounding-cell-cell-variation-due-to-cell-growth-in-single-cell-transcriptomics
#17
Thomas Blasi, Florian Buettner, Michael Strasser, Carsten Marr, Fabian Theis
MOTIVATION: Accessing gene expression at the single cell level has unraveled often large heterogeneity among seemingly homogeneous cells, which remained obscured in traditional population based approaches. The computational analysis of single-cell transcriptomics data, however, still imposes unresolved challenges with respect to normalization, visualization and modeling the data. One such issue are differences in cell size, which introduce additional variability into the data, for which appropriate normalization techniques are needed...
February 15, 2017: Physical Biology
https://www.readbyqxmd.com/read/28198180/engineering-recombinant-virus-like-nanoparticles-from-plants-for-cellular-delivery
#18
Lou Brillault, Philippe V Jutras, Noor Dashti, Eva C Thuenemann, Garry Morgan, George P Lomonossoff, Michael J Landsberg, Frank Sainsbury
Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification of virus-like nanoparticles for biomedical and nanotechnology applications. Here, we use plant-based transient expression of the Bluetongue virus (BTV) structural proteins, VP3 and VP7, to obtain high yields of empty and green fluorescent protein (GFP)-encapsidating core-like particles (CLPs) from leaves. Single-particle cryo-electron microscopy of both types of particles revealed considerable differences in CLP structure compared to the crystal structure of infection-derived CLPs; in contrast, the two recombinant CLPs have an identical external structure...
February 15, 2017: ACS Nano
https://www.readbyqxmd.com/read/28196962/spatial-and-temporal-analysis-of-alphavirus-replication-and-assembly-in-mammalian-and-mosquito-cells
#19
Joyce Jose, Aaron B Taylor, Richard J Kuhn
Sindbis virus (SINV [genus Alphavirus, family Togaviridae]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells...
February 14, 2017: MBio
https://www.readbyqxmd.com/read/28196667/stress-granules-assembly-affects-detection-of-mrna-in-living-cells-by-the-nanoflares-an-important-aspect-of-the-technology
#20
Magdalena Wolczyk, Paulina Podszywalow-Bartnicka, Lukasz Bugajski, Katarzyna Piwocka
The recently announced new methodologies to detect mRNA molecules in single cells offer opportunities for research, medicine and molecular diagnostics. The NanoFlare RNA Detection Probes are tools for characterizing RNA content (not localization) using fluorescence-based approaches in living cells. Combined with flow cytometry NanoFlares have expanded the available possibilities of quantitative analysis of mRNA level in a single cell. Herein we present, that the specific NanoFlare probes (SmartFlares), in some cases detect different amounts of mRNA compared to qPCR...
February 11, 2017: Biochimica et Biophysica Acta
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