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Single cell analysis

Chor Ho Jo, Sua Kim, Joon-Sung Park, Gheun-Ho Kim
BACKGROUND/AIMS: Dipeptidyl peptidase-4 (DPP4) inhibitors are known to have a protective effect on diabetic kidney disease, possibly via reduction of oxidative stress and inflammation in the kidney. However, whether these potential mechanisms play a role in non-diabetic proteinuric kidney diseases is not clear. METHODS: Two different animal experiments were carried out using sitagliptin and linagliptin for DPP4 inhibition. In each experiment, male Sprague-Dawley rats were uninephrectomized and randomly divided into vehicle-treated and doxorubicin-treated rats, with or without DPP4 inhibition...
June 15, 2018: Kidney & Blood Pressure Research
Prince Kumar Lat, Dipankar Sen
Massive expansion of a DNA hexanucleotide sequence repeat (C2G4) within the human C9orf72 gene has been linked to a number of neurodegenerative diseases. In sodium or potassium salt solutions, single-stranded d(C2G4)n DNAs fold to form G-quadruplexes. We have found that in magnesium or lithium salt solutions, especially under slightly acidic conditions, d(C2G4)n oligonucleotides fold to form a distinctive higher order structure whose most striking feature is an "inverted" circular dichroism spectrum, which is distinguishable from the spectrum of the left handed DNA double-helix, Z-DNA...
2018: PloS One
Kenneth D Harris, Hannah Hochgerner, Nathan G Skene, Lorenza Magno, Linda Katona, Carolina Bengtsson Gonzales, Peter Somogyi, Nicoletta Kessaris, Sten Linnarsson, Jens Hjerling-Leffler
Understanding any brain circuit will require a categorization of its constituent neurons. In hippocampal area CA1, at least 23 classes of GABAergic neuron have been proposed to date. However, this list may be incomplete; additionally, it is unclear whether discrete classes are sufficient to describe the diversity of cortical inhibitory neurons or whether continuous modes of variability are also required. We studied the transcriptomes of 3,663 CA1 inhibitory cells, revealing 10 major GABAergic groups that divided into 49 fine-scale clusters...
June 18, 2018: PLoS Biology
Cuiling Lu, Hongbin Chi, Yapeng Wang, Xue Feng, Lina Wang, Shuo Huang, Liying Yan, Shengli Lin, Ping Liu, Jie Qiao
In an attempt to explore the early developmental arrest in embryos from polycystic ovarian syndrome (PCOS) patients, we sequenced the transcriptome profiles of PCOS arrested 2-cell embryos, non-PCOS arrested 2-cell embryos and non-arrested 2-cell embryos using single-cell RNA-Seq technique. Differential expression analysis was performed using the DEGSeq R package. Gene Ontology (GO) enrichment was analyzed using the GOseq R package. Data revealed 62 differentially expressed genes between non-PCOS arrested and PCOS arrested embryos and 2217 differentially expressed genes between PCOS arrested and non-arrested 2-cell embryos...
June 18, 2018: Cell Cycle
Pascal Giehr, Charalampos Kyriakopoulos, Konstantin Lepikhov, Stefan Wallner, Verena Wolf, Jörn Walter
The controlled and stepwise oxidation of 5mC to 5hmC, 5fC and 5caC by Tet enzymes is influencing the chemical and biological properties of cytosine. Besides direct effects on gene regulation, oxidised forms influence the dynamics of demethylation and re-methylation processes. So far, no combined methods exist which allow to precisely determine the strand specific localisation of cytosine modifications along with their CpG symmetric distribution. Here we describe a comprehensive protocol combining conventional hairpin bisulfite with oxidative bisulfite sequencing (HPoxBS) to determine the strand specific distribution of 5mC and 5hmC at base resolution...
June 15, 2018: Nucleic Acids Research
E L Deters, R S Stokes, O N Genther-Schroeder, S L Hansen
To evaluate the effects of a Saccharomyces cerevisiae fermentation product (SCFP; Original XPC, Diamond V, Cedar Rapids, IA) on growth performance and antioxidant defense of newly weaned beef cattle, 180 single-source steers (278 ± 21 kg; SD) were used in a 56-d receiving study. Seven days after arrival, steers were blocked by body weight (BW) to pens of six and randomly assigned to treatments: SCFP at 0 (CON), 14 (SCFP14), or 28 (SCFP28) g·steer-1·d-1. Pen was the experimental unit (n = 10 per treatment)...
June 15, 2018: Journal of Animal Science
N Bourcigaux, C Rubino, I Berthaud, M E Toubert, B Donadille, L Leenhardt, I Petrot-Keller, S Brailly-Tabard, J Fromigué, F de Vathaire, T Simon, J P Siffroi, M Schlumberger, P Bouchard, S Christin-Maitre
STUDY QUESTION: What are the consequences of radioactive iodine (RAI) therapy for testicular function? SUMMARY ANSWER: A single activity of 3.7 GBq RAI for differentiated thyroid carcinoma (DTC) treatment in young men transiently altered Sertoli cell function and induced sperm chromosomal abnormalities. WHAT IS KNOWN ALREADY: Few studies, mainly retrospective, have reported the potential impacts of RAI on endocrine and exocrine testicular function...
June 15, 2018: Human Reproduction
Hector Guillen-Ahlers, Prahlad K Rao, Danu S Perumalla, Maria J Montoya, Avinash Y L Jadhav, Michael R Shortreed, Lloyd M Smith, Michael Olivier
The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods...
June 1, 2018: Journal of Visualized Experiments: JoVE
Caleb Sutherland, Yu Wang, Robert V Brown, Julie Foley, Beth Mahler, Kyathanahalli S Janardhan, Ramesh C Kovi, Anton M Jetten
Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma...
June 3, 2018: Journal of Visualized Experiments: JoVE
D P Jacobsen, A Moen, F Haugen, J Gjerstad
Aims Lumbar radicular pain following intervertebral disc herniation may be caused by a local inflammatory response induced by nucleus pulposus (NP) cells. Here in an animal model mimicking the clinical situation following disc herniation we investigated the effect of NP on the spinal nociceptive signaling and local gene expression. Methods In anaesthetized Lewis rats, extracellular single unit recordings of spinal nociceptive activity and qPCR were used to explore the effect of NP application onto the dorsal nerve roots (L3-L5)...
December 29, 2017: Scandinavian Journal of Pain
Felix R H Jonas, Kate E Royle, Rochelle Aw, Guy-Bart V Stan, Karen M Polizzi
Adaptation allows organisms to maintain a constant internal environment, which is optimised for growth. The unfolded protein response (UPR) is an example of a feedback loop that maintains endoplasmic reticulum (ER) homeostasis, and is characteristic of how adaptation is often mediated by transcriptional networks. The more recent discovery of asymmetric division in maintaining ER homeostasis, however, is an example of how alternative non-transcriptional pathways can exist, but are overlooked by gold standard transcriptomic or proteomic population-based assays...
March 2018: Synthetic and Systems Biotechnology
Ge-Min Fang, Jasmine Chamiolo, Svenja Kankowski, Felix Hövelmann, Dhana Friedrich, Alexander Löwer, Jochen C Meier, Oliver Seitz
Oligonucleotide probes that show enhanced fluorescence upon nucleic acid hybridization enable the detection and visualization of specific mRNA molecules, in vitro and in cellulo . A challenging problem is the analysis of single nucleotide alterations that occur, for example, when cellular mRNA is subject to C → U editing. Given the length required for uniqueness of the targeted segment, the commonly used probes do not provide the level of sequence specificity needed to discriminate single base mismatched hybridization...
June 7, 2018: Chemical Science
Gang Li, Mengyu Shen, Yuhui Yang, Shuai Le, Ming Li, Jing Wang, Yan Zhao, Yinling Tan, Fuquan Hu, Shuguang Lu
Adaptation of bacteria to phage predation poses a major obstacle for phage therapy. Bacteria adopt multiple mechanisms, such as inhibition of phage adsorption and CRISPR/Cas systems, to resist phage infection. Here, a phage-resistant mutant of Pseudomonas aeruginosa strain PA1 under the infection of lytic phage PaP1 was selected for further study. The PaP1-resistant variant, termed PA1RG, showed decreased adsorption to PaP1 and was devoid of long chain O -antigen on its cell envelope. Whole genome sequencing and comparative analysis revealed a single nucleotide mutation in the gene PA1S_08510, which encodes the O -antigen polymerase Wzy that is involved in lipopolysaccharide (LPS) biosynthesis...
2018: Frontiers in Microbiology
A Alshangiti, G Chandhoke, P M Ellis
Angiogenesis is frequent in non-small-cell lung cancer (nsclc) and is associated with more aggressive disease. Many clinical trials have evaluated the addition of antiangiogenic therapy to standard therapies for patients with nsclc. Bevacizumab, a monoclonal antibody directed against serum vascular endothelial growth factor, in combination with carboplatin-paclitaxel chemotherapy, has been shown to improve survival for patients with nsclc. However, bevacizumab-based therapy is not suitable for many nsclc patients, including those with squamous histology, poor performance status, brain metastases, and the presence of bleeding or thrombotic disorders...
June 2018: Current Oncology
Kristofer Davie, Jasper Janssens, Duygu Koldere, Maxime De Waegeneer, Uli Pech, Łukasz Kreft, Sara Aibar, Samira Makhzami, Valerie Christiaens, Carmen Bravo González-Blas, Suresh Poovathingal, Gert Hulselmans, Katina I Spanier, Thomas Moerman, Bram Vanspauwen, Sarah Geurs, Thierry Voet, Jeroen Lammertyn, Bernard Thienpont, Sha Liu, Nikos Konstantinides, Mark Fiers, Patrik Verstreken, Stein Aerts
The diversity of cell types and regulatory states in the brain, and how these change during aging, remains largely unknown. We present a single-cell transcriptome atlas of the entire adult Drosophila melanogaster brain sampled across its lifespan. Cell clustering identified 87 initial cell clusters that are further subclustered and validated by targeted cell-sorting. Our data show high granularity and identify a wide range of cell types. Gene network analyses using SCENIC revealed regulatory heterogeneity linked to energy consumption...
June 9, 2018: Cell
Andre du Toit, Jan-Hendrik S Hofmeyr, Thomas J Gniadek, Ben Loos
Macroautophagy/autophagy is a proteolytic pathway that is involved in both bulk degradation of cytoplasmic proteins as well as in selective degradation of cytoplasmic organelles. Autophagic flux is often defined as a measure of autophagic degradation activity, and many techniques exist to assess autophagic flux. Although these techniques have generated invaluable information about the autophagic system, the quest continues for developing methods that not only enhance sensitivity and provide a means of quantification, but also accurately reflect the dynamic character of the pathway...
June 17, 2018: Autophagy
Leighton H Daigh, Chad Liu, Mingyu Chung, Karlene A Cimprich, Tobias Meyer
Faithful DNA replication is challenged by stalling of replication forks during S phase. Replication stress is further increased in cancer cells or in response to genotoxic insults. Using live single-cell image analysis, we found that CDK2 activity fluctuates throughout an unperturbed S phase. We show that CDK2 fluctuations result from transient ATR signals triggered by stochastic replication stress events. In turn, fluctuating endogenous CDK2 activity causes corresponding decreases and increases in DNA synthesis rates, linking changes in stochastic replication stress to fluctuating global DNA replication rates throughout S phase...
June 4, 2018: Cell Systems
Andrew G Diamos, Hugh S Mason
Recombinant virus-like particles (VLPs) are proven to be safe and effective vaccine candidates. We have previously described a plant-based recombinant protein expression system based on agroinfiltration of a replicating vector derived from the geminivirus bean yellow dwarf virus (BeYDV). The system has been systematically optimized to improve expression and reduce cell death in Nicotiana benthamiana leaves. Using these modifications, we show that VLPs derived from genotype GII.4 norovirus, the leading cause of acute gastroenteritis worldwide, can be produced at >1 mg/g leaf fresh weight (LFW), over three times the highest level ever reported in plant-based systems...
June 14, 2018: Protein Expression and Purification
Geert Rb Huys, Jeroen Raes
With the vast majority of the microbial world still considered unculturable or undiscovered, microbiologists not only require more fundamental insights concerning microbial growth requirements but also need to implement miniaturized, versatile and high-throughput technologies to upscale current microbial isolation strategies. In this respect, single-cell-based approaches are increasingly finding their way to the microbiology lab. A number of recent studies have demonstrated that analysis and separation of free microbial cells by flow-based sorting as well as physical stochastic confinement of individual cells in microenvironment compartments can facilitate the isolation of previously uncultured species and the discovery of novel microbial taxa...
June 13, 2018: Current Opinion in Microbiology
Gro E K Bjerga, Øivind Larsen, Hasan Arsın, Adele Williamson, Antonio García-Moyano, Ingar Leiros, Pål Puntervoll
Intracellular subtilisin proteases (ISPs) have important roles in protein processing during the stationary phase in bacteria. Their unregulated protein degrading activity may have adverse effects inside a cell, but little is known about their regulatory mechanism. Until now, ISPs have mostly been described from Bacillus species, with structural data from a single homolog. Here, we study a marine ISP originating from a phylogenetically distinct genus, Planococcus sp. The enzyme was successfully overexpressed in E...
June 16, 2018: Proteins
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