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Vid Leko, Smitha Sripathy, Robin L Adrianse, Taylor Loe, Angela Park, Uyen Lao, Eric J Foss, Marisa S Bartolomei, Antonio Bedalov
Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi)...
March 2, 2018: Journal of Visualized Experiments: JoVE
E Savi, S Peveri, C Cavaliere, S Masieri, M Montagni
The introduction of highly purified natural and recombinant single allergenic molecules represented an important improvement in the diagnosis of IgE sensitization. The identification of specific IgE against cross-reacting molecules such as profilin, lipid transfer proteins, calcium binding proteins or against “genuine molecules”, represents an added value and allows to distinguish between true and false polysensitization. In vitro tests add information to recognize patients with sensitization to genuine molecules that cause allergic diseases and to evaluate in childhood the spreading of sensitization for each molecule in order to choose the best treatment and to identify the ideal patient for allergen immunotherapy...
January 2018: Journal of Biological Regulators and Homeostatic Agents
Hussaina Banu, N Renuka, S M Faheem, Raees Ismail, Vinita Singh, Zahra Saadatmand, Saad Sultan Khan, Kavya Narayanan, Alma Raheem, Kumpati Premkumar, Geetha Vasanthakumar
Recently, several attempts have been made to use the phytopharmaceuticals from plant extracts as reducing, capping and stabilizing agents for the biomimetic synthesis of various metal nanoparticles conjugated to the phytopharmaceuticals. These biogenic metal nanoparticles are non-toxic and can be used as contrast agents, drug delivery vehicles and photothermal agents for cancer therapy. Herein, we report the synthesis of both silver and gold nanoparticles using the pollen extract of Phoenix dactylifera (Date Palm), characterization using UV-visible spectroscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy, quantitation of phytochemicals capping the nanoparticles using Folin - Ciocalteu's method, cytotoxicity studies on MCF-7 breast cancer cells, cancer cell death analysis using fluorescent microscopy, and modulation of expression of the pro-apoptotic p53 and anti-apoptotic Bcl-2 proteins...
March 18, 2018: Biological Trace Element Research
Sunil Ghatge, Youri Yang, Woo-Young Song, Tae-Young Kim, Hor-Gil Hur
In the present study, the gene encoding a multicopper oxidase, more precisely a laccase from the thermoalkaliphilic aerobic bacterium Caldalkalibacillus thermarum strain TA2.A1 (CtLac), was cloned and expressed in Escherichia coli. CtLac is a monomeric protein with a molecular weight of 57 kDa as determined by native polyacrylamide gel electrophoresis. The optimum pH and temperature for 2,6-dimethoxyphenol (2,6-DMP) oxidation were 8.0 and 70 °C, respectively. The kinetic constants Km and kcat for 2,6-DMP were of 200 μM and 23 s-1 , respectively...
March 19, 2018: Applied Microbiology and Biotechnology
Anna J Komor, Andrew J Jasniewski, Lawrence Que, John D Lipscomb
Covering: up to 2017The participation of non-heme dinuclear iron cluster-containing monooxygenases in natural product biosynthetic pathways has been recognized only recently. At present, two families have been discovered. The archetypal member of the first family, CmlA, catalyzes β-hydroxylation of l-p-aminophenylalanine (l-PAPA) covalently linked to the nonribosomal peptide synthetase (NRPS) CmlP, thereby effecting the first step in the biosynthesis of chloramphenicol by Streptomyces venezuelae. CmlA houses the diiron cluster in a metallo-β-lactamase protein fold instead of the 4-helix bundle fold of nearly every other diiron monooxygenase...
March 19, 2018: Natural Product Reports
S Junaid S Qazi, Raymond J Turner
EmrE is a member of the small multidrug resistance (SMR) protein family in Escherichia coli . It confers resistance to a wide variety of quaternary cation compounds (QCCs) as an efflux transporter driven by the transmembrane proton motive force. We have expressed hexahistidinyl (His6 ) - myc epitope tagged EmrE, extracted it from membrane preparations using the detergent n-dodecyl-β-D-maltopyranoside (DDM), and purified it using nickel-affinity chromatography. The size of the EmrE protein, in DDM environment, was then examined in the presence and absence of a range of structurally different QCC ligands that varied in their chemical structure, charge and shape...
March 2018: Biochemistry and Biophysics Reports
Shweta Duggal, Noor Jailkhani, Mukul Kumar Midha, Kanury V S Rao, Ajay Kumar
The Rb protein is a tumor suppressor protein that regulates the key G1S checkpoint consequently blocking the progression of cell cycle into S-phase. Despite its pertinent role in cell cycle regulation, comprehensive information on its interacting partners across cell cycle progression is lacking. Here, we aim to submit a comprehensive set of Rb interactors as the cell progresses from G0 through G1 and S into G2 phase in HEK 293 cell line. Affinity purification of HA-tagged Rb protein along with its interactors was analyzed by mass spectrometry (AP-MS)...
April 2018: Data in Brief
Seeyoung Choi, Paul E Love
Oxidative inactivation of cysteine-dependent Protein Tyrosine Phosphatases (PTPs) by cellular reactive oxygen species (ROS) plays a critical role in regulating signal transduction in multiple cell types. The phosphatase activity of most PTPs depends upon a 'signature' cysteine residue within the catalytic domain that is maintained in the de-protonated state at physiological pH rendering it susceptible to ROS-mediated oxidation. Direct and indirect techniques for detection of PTP oxidation have been developed (Karisch and Neel, 2013)...
January 5, 2018: Bio-protocol
Emily Tubman, Yungui He, Thomas S Hays, David J Odde
Introduction: The microtubule motor protein kinesin-5 is well known to establish the bipolar spindle by outward sliding of antiparallel interpolar microtubules. In yeast, kinesin-5 also facilitates chromosome alignment "congression" at the spindle equator by preferentially depolymerizing long kinetochore microtubules (kMTs). The motor protein kinesin-8 has also been linked to chromosome congression. Therefore, we sought to determine whether kinesin-5 or kinesin-8 facilitates chromosome congression in insect spindles...
February 2018: Cellular and Molecular Bioengineering
Chunyan Mo, Shumin Wan, Youquan Xia, Ning Ren, Yang Zhou, Xingyu Jiang
Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL ( MeCBL ) and 26 CIPK ( MeCIPK ) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBL s and CIPK s. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle...
2018: Frontiers in Plant Science
Chao Shi, Bei-Qing Pan, Feng Shi, Zhi-Hui Xie, Yan-Yi Jiang, Li Shang, Yu Zhang, Xin Xu, Yan Cai, Jia-Jie Hao, Ming-Rong Wang
Esophageal squamous cell carcinoma (ESCC) is one of the malignancies in digestive system, with a low 5-year survival rate. We previously revealed that Sequestosome 1 (SQSTM1/p62) protein levels were upregulated in ESCC tissues. However, it is unclear about the function of p62 and the underlying mechanism. Here, we used immunofluorescence and immunohistochemistry to investigate the expression of p62 in ESCC. Western blotting, quantitative RT-PCR, colony formation assay, flow cytometry, immunoprecipitation and xenograft tumor assay were used to analyze the role of p62 in vitro and vivo...
March 19, 2018: Oncogene
Caslin A Gilroy, Stefan Roberts, Ashutosh Chilkoti
Fibroblast growth factor 21 (FGF21) is under investigation as a type 2 diabetes protein drug, but its efficacy is impeded by rapid in vivo clearance and by costly production methods. To improve the protein's therapeutic utility, we recombinantly expressed FGF21 as a fusion with an elastin-like polypeptide (ELP), a peptide polymer that exhibits reversible thermal phase behavior. Below a critical temperature, ELPs exist as miscible unimers, while above, they associate into a coacervate. The thermal responsiveness of ELPs is retained upon fusion to proteins, which has notable consequences for the production and in vivo delivery of FGF21...
March 15, 2018: Journal of Controlled Release: Official Journal of the Controlled Release Society
Anne L Carlton, Anuradha Illendula, Yan Gao, Danielle C Llaneza, Adam Boulton, Anant Shah, Roger A Rajewski, Charles N Landen, David Wotton, John H Bushweller
OBJECTIVE: Ovarian cancer survival and treatment have improved minimally in the past 20years. Novel treatment strategies are needed to combat this disease. This study investigates the effects of chemical inhibition of the CBFβ/RUNX protein-protein interaction on ovarian cancer cell lines. METHODS: Ovarian cancer cell lines were treated with CBFβ/RUNX inhibitors, and the effects on proliferation, DNA replication, wound healing, and anchorage-independent growth were measured...
March 15, 2018: Gynecologic Oncology
Winston X Yan, Shaorong Chong, Huaibin Zhang, Kira S Makarova, Eugene V Koonin, David R Cheng, David A Scott
Bacterial class 2 CRISPR-Cas systems utilize a single RNA-guided protein effector to mitigate viral infection. We aggregated genomic data from multiple sources and constructed an expanded database of predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D, encoding dual HEPN domain-containing Cas13d effectors and putative WYL-domain-containing accessory proteins (WYL1 and WYL-b1 through WYL-b5). The median size of Cas13d proteins is 190 to 300 aa smaller than that of Cas13a-Cas13c...
March 9, 2018: Molecular Cell
Magdalena Martinez-Losa, Tara E Tracy, Keran Ma, Laure Verret, Alexandra Clemente-Perez, Abdullah S Khan, Inma Cobos, Kaitlyn Ho, Li Gan, Lennart Mucke, Manuel Alvarez-Dolado, Jorge J Palop
Inhibitory interneurons regulate the oscillatory rhythms and network synchrony that are required for cognitive functions and disrupted in Alzheimer's disease (AD). Network dysrhythmias in AD and multiple neuropsychiatric disorders are associated with hypofunction of Nav1.1, a voltage-gated sodium channel subunit predominantly expressed in interneurons. We show that Nav1.1-overexpressing, but not wild-type, interneuron transplants derived from the embryonic medial ganglionic eminence (MGE) enhance behavior-dependent gamma oscillatory activity, reduce network hypersynchrony, and improve cognitive functions in human amyloid precursor protein (hAPP)-transgenic mice, which simulate key aspects of AD...
March 15, 2018: Neuron
Travis R Helgren, Elif S Seven, Congling Chen, Thomas E Edwards, Bart L Staker, Jan Abendroth, Peter J Myler, James R Horn, Timothy J Hagen
Methionine aminopeptidase (MetAP) is a dinuclear metalloprotease responsible for the cleavage of methionine initiator residues from nascent proteins. MetAP activity is necessary for bacterial proliferation and is therefore a projected novel antibacterial target. A compound library consisting of 294 members containing metal-binding functional groups was screened against Rickettsia prowazekii MetAP to determine potential inhibitory motifs. The compounds were first screened against the target at a concentration of 10 µM and potential hits were determined to be those exhibiting greater than 50% inhibition of enzymatic activity...
March 15, 2018: Bioorganic & Medicinal Chemistry Letters
Mauro E M Nunes, Talise E Müller, Camila Murussi, Aline M B Amaral, Jeane L C Gomes, Aline T Marins, Jossiele Leitemperger, Cintia C R Rodrigues, Tiago L Fiuza, Maiara D Costa, Eduardo S Severo, Denis B Rosemberg, Vania L Loro
The use of commercial pesticides combinations increases the risk of intoxication in non-target aquatic organisms. Here, we investigate the potential of a commercial pesticide formulation containing (CYP) plus chlopyrifos (CPF) to induce oxidative damage on two fish species (common carp and zebrafish). Carp and zebrafish were exposed for 96 h under laboratory conditions. Fish were divided in three different groups: CTL, 0.3 μg L-1 or 0.6 μg L-1 of CYP and 0.5 or 1 μg L-1 of CPF in commercial formulation...
March 15, 2018: Comparative Biochemistry and Physiology. Toxicology & Pharmacology: CBP
Rajiv K Khajuria, Mathias Munschauer, Jacob C Ulirsch, Claudia Fiorini, Leif S Ludwig, Sean K McFarland, Nour J Abdulhay, Harrison Specht, Hasmik Keshishian, D R Mani, Marko Jovanovic, Steven R Ellis, Charles P Fulco, Jesse M Engreitz, Sabina Schütz, John Lian, Karen W Gripp, Olga K Weinberg, Geraldine S Pinkus, Lee Gehrke, Aviv Regev, Eric S Lander, Hanna T Gazda, Winston Y Lee, Vikram G Panse, Steven A Carr, Vijay G Sankaran
Blood cell formation is classically thought to occur through a hierarchical differentiation process, although recent studies have shown that lineage commitment may occur earlier in hematopoietic stem and progenitor cells (HSPCs). The relevance to human blood diseases and the underlying regulation of these refined models remain poorly understood. By studying a genetic blood disorder, Diamond-Blackfan anemia (DBA), where the majority of mutations affect ribosomal proteins and the erythroid lineage is selectively perturbed, we are able to gain mechanistic insight into how lineage commitment is programmed normally and disrupted in disease...
March 9, 2018: Cell
Danica M Sutherland, Pavithra Aravamudhan, Terence S Dermody
Viruses are constantly engaged in a molecular arms race with the host, where efficient and tactical use of cellular receptors benefits critical steps in infection. Receptor use dictates initiation, establishment, and spread of viral infection to new tissues and hosts. Mammalian orthoreoviruses (reoviruses) are pervasive pathogens that use multiple receptors to overcome protective host barriers to disseminate from sites of initial infection and cause disease in young mammals. In particular, reovirus invades the central nervous system (CNS) with serotype-dependent tropism and disease...
2018: Advances in Virus Research
Zi-Yi Li, Min Huang, Xiu-Kun Wang, Ying Zhu, Jin-Song Li, Catherine Cl Wong, Qun Fang
Single cell proteomic analysis provides crucial information on cellular heterogeneity in biological systems. Herein, we describe a nanoliter-scale oil-air-droplet (OAD) chip for achieving multi-step complex sample pretreatment and injection for single cell proteomic analysis under the shotgun mode. By using miniaturized stationary droplet microreaction and manipulation techniques, our system allows all sample pretreatment and injection procedures to be performed in a nanoliter-scale droplet with minimum sample loss and a high sample injection efficiency (> 99%), thus substantially increasing the analysis sensitivity for single cell samples...
March 19, 2018: Analytical Chemistry
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