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A Role for Mediator Core in Limiting Coactivator Recruitment in Saccharomyces cerevisiae.

Genetics 2020 Jun;215(2):407-420

Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah 84112.

Mediator is an essential, multisubunit complex that functions as a transcriptional coactivator in yeast and other eukaryotic organisms. Mediator has four conserved modules, Head, Middle, Tail, and Kinase, and has been implicated in nearly all aspects of gene regulation. The Tail module has been shown to recruit the Mediator complex to the enhancer or upstream activating sequence (UAS) regions of genes via interactions with transcription factors, and the Kinase module facilitates the transition of Mediator from the UAS/enhancer to the preinitiation complex via protein phosphorylation. Read More

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An assay of human tyrosine protein kinase ABL activity using an protein expression system.

Biotechniques 2021 Apr 6;70(4):209-217. Epub 2021 Apr 6.

Department of Functional Molecular Science, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 7348553, Japan.

ABL, a human tyrosine protein kinase, and its substrate are co-expressed in . Tyrosine phosphorylation of the substrate in was detected using Phos-tag SDS-PAGE. The bacterial co-expression system was used as a field for the kinase reaction to evaluate the enzymatic activity of five types of ABL kinase domain mutants. Read More

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Ddx41 inhibition of DNA damage signaling permits erythroid progenitor expansion in zebrafish.

Haematologica 2021 Mar 25. Epub 2021 Mar 25.

Albert Einstein College of Medicine, Department of Developmental and Molecular Biology, Bronx, NY, USA; Albert Einstein College of Medicine, Gottesman Institute for Stem Cell Biology and Regenerative Medicine, Bronx, NY, USA; Albert Einstein College of Medicine and Montefiore Medical Center, Department of Medicine (Oncology), Bronx, NY.

DEAD-box Helicase 41 (DDX41) is a recently identified factor mutated in hematologic malignancies whose function in hematopoiesis is unknown. Using an in vivo model of Ddx41 deficiency, we unveiled a critical role for this helicase in regulating erythropoiesis. We demonstrated that loss of ddx41 leads to anemia caused by diminished proliferation and defective differentiation of erythroid progenitors. Read More

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Agar Invasion Assays.

Bio Protoc 2020 Aug 20;10(16):e3730. Epub 2020 Aug 20.

Department of Microbiology and Immunology, Stony Brook University, Stony Brook, NY, USA.

The ability of the human fungal pathogen to disseminate into tissues is promoted by a switch from budding to invasive hyphal growth. This morphological transition is stimulated by multiple environmental factors that can vary at different sites of infection. To identify genes that promote invasive growth, mutants can be screened for defects in growing invasively into solid agar medium as a substitute for studying tissue invasion. Read More

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The lipoprotein NlpD in responds to acid stress and regulates macrophage resistance and virulence by maintaining membrane integrity.

Virulence 2021 12;12(1):415-429

Tianjin Key Laboratory of Food Science and Health, School of Medicine, Nankai University , Tianjin, China.

, an emerging opportunistic pathogen, is implicated in severe foodborne outbreak infections in premature and full-term infants. Generally, acid tolerance is vital for the pathogenesis of foodborne pathogens; however, its role in virulence remains largely unknown. To screen out acid-tolerance determinants from transposon mutants, anovel counterselection method using gentamicin and acid was developed. Read More

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December 2021

BED domain-containing NLR from wild barley confers resistance to leaf rust.

Plant Biotechnol J 2021 Jan 8. Epub 2021 Jan 8.

Plant Breeding Institute, The University of Sydney, Cobbitty, NSW, Australia.

Leaf rust, caused by Puccinia hordei, is a devastating fungal disease affecting barley (Hordeum vulgare subsp. vulgare) production globally. Despite the effectiveness of genetic resistance, the deployment of single genes often compromises durability due to the emergence of virulent P. Read More

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January 2021

Simultaneous silencing of two different Arabidopsis genes with a novel virus-induced gene silencing vector.

Plant Methods 2021 Jan 6;17(1). Epub 2021 Jan 6.

Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Haikou, 571101, China.

Background: Virus-induced gene silencing (VIGS) is a useful tool for functional characterizations of plant genes. However, the penetrance of VIGS varies depending on the genes to be silenced, and has to be evaluated by examining the transcript levels of target genes.

Results: In this report, we report the development of a novel VIGS vector that permits a preliminary assessment of the silencing penetrance. Read More

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January 2021

Mild Oxidative Stress Reduces NRF2 SUMOylation to Promote // Mutant Lung Adenocarcinoma Cell Migration and Invasion.

Oxid Med Cell Longev 2020 24;2020:6240125. Epub 2020 Nov 24.

Department of Biochemistry & Molecular Cell Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a crucial transcription factor for cell adaptation and defense against oxidative stress. NRF2 activation confers // (KLK) mutant tumor cells with greater resistance to oxidative insults. We previously reported that SUMOylation at lysine residue 110 is important for the ability of NRF2 to promote reactive oxygen species (ROS) clearance in hepatocellular carcinoma. Read More

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November 2020

The Host Cell Metabolite Inositol Hexakisphosphate Promotes Efficient Endogenous HIV-1 Reverse Transcription by Stabilizing the Viral Capsid.

mBio 2020 12 1;11(6). Epub 2020 Dec 1.

Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA

A defining activity of retroviruses is reverse transcription, the process by which the viral genomic RNA is converted into the double-stranded DNA required for virus replication. Reverse transcriptase (RT), the viral enzyme responsible for this process, was identified in 1970 by assaying permeabilized retrovirus particles for DNA synthesis Such reactions are inefficient, with only a small fraction of viral genomes being converted to full-length double-stranded DNA molecules, possibly owing to disruption of the structure of the viral core. Here, we show that reverse transcription in purified HIV-1 cores is enhanced by the addition of the capsid-binding host cell metabolite inositol hexakisphosphate (IP6). Read More

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December 2020

Vesicular trafficking permits evasion of cGAS/STING surveillance during initial human papillomavirus infection.

PLoS Pathog 2020 11 30;16(11):e1009028. Epub 2020 Nov 30.

Cancer Biology Graduate Interdisciplinary Program, The University of Arizona, Tucson, Arizona, United States of America.

Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. Read More

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November 2020

Double trouble: Bacillus depends on a functional Tat machinery to avoid severe oxidative stress and starvation upon entry into a NaCl-depleted environment.

Biochim Biophys Acta Mol Cell Res 2021 02 25;1868(2):118914. Epub 2020 Nov 25.

University of Groningen, University Medical Center Groningen, Department of Medical Microbiology, Groningen, the Netherlands. Electronic address:

The widely conserved twin-arginine translocases (Tat) allow the transport of fully folded cofactor-containing proteins across biological membranes. In doing so, these translocases serve different biological functions ranging from energy conversion to cell division. In the Gram-positive soil bacterium Bacillus subtilis, the Tat machinery is essential for effective growth in media lacking iron or NaCl. Read More

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February 2021

AUTOPHAGY-RELATED14 and Its Associated Phosphatidylinositol 3-Kinase Complex Promote Autophagy in Arabidopsis.

Plant Cell 2020 12 30;32(12):3939-3960. Epub 2020 Sep 30.

Department of Biology, Washington University in St. Louis, St. Louis, Missouri 63130

Phosphatidylinositol 3-phosphate (PI3P) is an essential membrane signature for both autophagy and endosomal sorting that is synthesized in plants by the class III phosphatidylinositol 3-kinase (PI3K) complex, consisting of the VPS34 kinase, together with ATG6, VPS15, and either VPS38 or ATG14 as the fourth subunit. Although Arabidopsis () plants missing the three core subunits are infertile, mutants are viable but have aberrant leaf, root, and seed development, Suc sensing, and endosomal trafficking, suggesting that VPS38 and ATG14 are nonredundant. Here, we evaluated the role of ATG14 through a collection of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and T-DNA insertion mutants disrupting the two Arabidopsis paralogs. Read More

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December 2020

Chromosome separation during Drosophila male meiosis I requires separase-mediated cleavage of the homolog conjunction protein UNO.

PLoS Genet 2020 10 1;16(10):e1008928. Epub 2020 Oct 1.

Department of Molecular Life Science (DMLS), University of Zurich, Zurich, Switzerland.

Regular chromosome segregation during the first meiotic division requires prior pairing of homologous chromosomes into bivalents. During canonical meiosis, linkage between homologous chromosomes is maintained until late metaphase I by chiasmata resulting from meiotic recombination in combination with distal sister chromatid cohesion. Separase-mediated elimination of cohesin from chromosome arms at the end of metaphase I permits terminalization of chiasmata and homolog segregation to opposite spindle poles during anaphase I. Read More

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October 2020

Broadly Neutralizing Antibodies for Influenza: Passive Immunotherapy and Intranasal Vaccination.

Vaccines (Basel) 2020 Jul 29;8(3). Epub 2020 Jul 29.

Department of Microbiology and Immunology, School of Medicine, Aichi Medical University, Aichi 480-1195, Japan.

Influenza viruses cause annual epidemics and occasional pandemics. The high diversity of viral envelope proteins permits viruses to escape host immunity. Therefore, the development of a universal vaccine and broadly neutralizing antibodies (bnAbs) is essential for controlling various mutant viruses. Read More

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Targeting translational read-through of premature termination mutations in with PTC124 for pulmonary arterial hypertension.

Pulm Circ 2020 Jul-Sep;10(3):2045894020935783. Epub 2020 Jul 20.

Department of Medicine, University of Cambridge School of Clinical Medicine, Cambridge, UK.

Pulmonary arterial hypertension is a fatal disorder of the lung circulation in which accumulation of vascular cells progressively obliterates the pulmonary arterioles. This results in sustained elevation in pulmonary artery pressure leading eventually to right heart failure. Approximately, 80% of familial and 20% of sporadic idiopathic pulmonary arterial hypertension cases are caused by mutations in the bone morphogenetic protein receptor type 2 (BMPR2). Read More

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Sequential Phosphorylation of the Hepatitis C Virus NS5A Protein Depends on NS3-Mediated Autocleavage between NS3 and NS4A.

J Virol 2020 09 15;94(19). Epub 2020 Sep 15.

Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan

Replication of the genotype 2 hepatitis C virus (HCV) requires hyperphosphorylation of the nonstructural protein NS5A. It has been known that NS5A hyperphosphorylation results from the phosphorylation of a cluster of highly conserved serine residues (S2201, S2208, S2211, and S2214) in a sequential manner. It has also been known that NS5A hyperphosphorylation requires an NS3 protease encoded on one single NS3-5A polyprotein. Read More

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September 2020

Assessing Budding Yeast Phosphoproteome Dynamics in a Time-Resolved Manner using TMT10plex Mass Tag Labeling.

STAR Protoc 2020 Jun 19;1(1):100022. Epub 2020 Jun 19.

Chromosome Segregation Laboratory, The Francis Crick Institute, London NW1 1AT, UK.

Amine-reactive Tandem Mass Tag 10plex (TMT10plex) labeling permits multiplexed protein identification and quantitative analysis by tandem mass spectrometry (MS/MS). We have used this technology to label 20 samples collected in a time-resolved manner from a wild-type and phosphatase mutant background to characterize phosphoproteome dynamics. Here, we provide a detailed protocol for biological and mass spectrometry sample preparation and analysis. Read More

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Light-Inducible CRISPR Labeling.

Methods Mol Biol 2020 ;2173:137-150

Synthetic Biology Group, BioQuant Center, University of Heidelberg, Heidelberg, Germany.

CRISPR labeling is a powerful technique to study the chromatin architecture in live cells. In CRISPR labeling, a catalytically dead CRISPR-Cas9 mutant is employed as programmable DNA-binding domain to recruit fluorescent proteins to selected genomic loci. The fluorescently labeled loci can then be identified as fluorescent spots and tracked over time by microscopy. Read More

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BRAF inhibition in melanoma is associated with the dysregulation of histone methylation and histone methyltransferases.

Neoplasia 2020 09 3;22(9):376-389. Epub 2020 Jul 3.

The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912, USA; Masonic Cancer Center, 2231 6th St SE, Minneapolis, MN 5545, USA. Electronic address:

The development of mutant BRAF inhibitors has improved the outcome for melanoma patients with BRAF mutations. Although the initial response to these inhibitors can be dramatic, sometimes resulting in complete tumor regression, the majority of melanomas become resistant. To study resistance to BRAF inhibition, we developed a novel mouse model of melanoma using a tetracycline/doxycycline-regulated system that permits control of mutant BRAF expression. Read More

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September 2020

Rapid, Seamless Generation of Recombinant Poxviruses using Host Range and Visual Selection.

J Vis Exp 2020 05 24(159). Epub 2020 May 24.

Department of Medial Microbiology and Immunology, School of Medicine, University of California Davis;

Vaccinia virus (VACV) was instrumental in eradicating variola virus (VARV), the causative agent of smallpox, from nature. Since its first use as a vaccine, VACV has been developed as a vector for therapeutic vaccines and as an oncolytic virus. These applications take advantage of VACV's easily manipulated genome and broad host range as an outstanding platform to generate recombinant viruses with a variety of therapeutic applications. Read More

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Measurements of Innate Immune Function in C. elegans.

Methods Mol Biol 2020 ;2144:145-160

Program in Innate Immunity, Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA, USA.

The microscopic nematode Caenorhabditis elegans has emerged as a powerful system to characterize evolutionarily ancient mechanisms of pathogen sensing, innate immune activation, and protective host responses. Experimentally, C. elegans can be infected with a wide variety of human pathogens, as well as with natural pathogens of worms that were isolated from wild-caught nematodes. Read More

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Selective Degradation Permits a Feedback Loop Controlling Annexin A6 and Cholesterol Levels in Endolysosomes of NPC1 Mutant Cells.

Cells 2020 05 7;9(5). Epub 2020 May 7.

Departament de Biomedicina, Unitat de Biologia Cel·lular, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036 Barcelona, Spain.

We recently identified elevated annexin A6 (AnxA6) protein levels in Niemann-Pick-type C1 (NPC1) mutant cells. In these cells, AnxA6 depletion rescued the cholesterol accumulation associated with NPC1 deficiency. Here, we demonstrate that elevated AnxA6 protein levels in NPC1 mutants or upon pharmacological NPC1 inhibition, using U18666A, were not due to upregulated AnxA6 mRNA expression, but caused by defects in AnxA6 protein degradation. Read More

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A Role for Mediator Core in Limiting Coactivator Recruitment in .

Genetics 2020 06 23;215(2):407-420. Epub 2020 Apr 23.

Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah 84112

Mediator is an essential, multisubunit complex that functions as a transcriptional coactivator in yeast and other eukaryotic organisms. Mediator has four conserved modules, Head, Middle, Tail, and Kinase, and has been implicated in nearly all aspects of gene regulation. The Tail module has been shown to recruit the Mediator complex to the enhancer or upstream activating sequence (UAS) regions of genes via interactions with transcription factors, and the Kinase module facilitates the transition of Mediator from the UAS/enhancer to the preinitiation complex via protein phosphorylation. Read More

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Understanding Lineage Plasticity as a Path to Targeted Therapy Failure in -Mutant Non-small Cell Lung Cancer.

Front Genet 2020 27;11:281. Epub 2020 Mar 27.

Department of Pharmacology and Therapeutics, Roswell Park Comprehensive Cancer Center, Buffalo, NY, United States.

Somatic alterations in the epidermal growth factor receptor gene () result in aberrant activation of kinase signaling and occur in ∼15% of non-small cell lung cancers (NSCLC). Patients diagnosed with -mutant NSCLC have good initial clinical response to EGFR tyrosine kinase inhibitors (EGFR TKIs), yet tumor recurrence is common and quick to develop. Mechanisms of acquired resistance to EGFR TKIs have been studied extensively over the past decade. Read More

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Single-cell analysis of a mutant library generated using CRISPR-guided deaminase in human melanoma cells.

Commun Biol 2020 04 2;3(1):154. Epub 2020 Apr 2.

Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea.

CRISPR-based screening methods using single-cell RNA sequencing (scRNA-seq) technology enable comprehensive profiling of gene perturbations from knock-out mutations. However, evaluating substitution mutations using scRNA-seq is currently limited. We combined CRISPR RNA-guided deaminase and scRNA-seq technology to develop a platform for introducing mutations in multiple genes and assessing the mutation-associated signatures. Read More

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SLAM-MS: Mutation scanning of stem-loop amplicons with TaqMan probes by quantitative DNA melting analysis.

Sci Rep 2020 03 25;10(1):5476. Epub 2020 Mar 25.

N.N. Blokhin Russian Cancer Research Center, Ministry of Health of Russia, 24 Kashirskoye Shosse, Moscow, 115478, Russia.

DNA Melting Analysis (DMA) with a TaqMan probe covering the mutation "hot spot" is a simple, sensitive, and "closed tube" method of mutation detection. However, DMA requires asymmetric PCR to produce single-stranded amplicons capable of interacting with TaqMan probes. This makes quantitative analysis impossible owing to low amplification efficiency. Read More

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Schnyder corneal dystrophy-associated UBIAD1 is defective in MK-4 synthesis and resists autophagy-mediated degradation.

J Lipid Res 2020 05 18;61(5):746-757. Epub 2020 Mar 18.

Departments of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046

The autosomal dominant disorder Schnyder corneal dystrophy (SCD) is caused by mutations in UbiA prenyltransferase domain-containing protein-1 (UBIAD1), which uses geranylgeranyl pyrophosphate (GGpp) to synthesize the vitamin K subtype menaquinone-4 (MK-4). SCD is characterized by opacification of the cornea, owing to aberrant build-up of cholesterol in the tissue. We previously discovered that sterols stimulate association of UBIAD1 with ER-localized HMG-CoA reductase, which catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids, including GGpp. Read More

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Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting.

J Vis Exp 2020 01 29(155). Epub 2020 Jan 29.

Department of Molecular, Cellular, and Developmental Biology, Yale University;

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as axolotls, to regenerate entire limbs as adults. Here, we outline techniques used to generate chimeric axolotls with Cas9-mutagenized haploid forelimbs that can be used for exploring gene function and the fidelity of limb regeneration. We combine several embryological and genetic techniques, including haploid generation via in vitro activation, CRISPR/Cas9 mutagenesis, and tissue grafting into one protocol to produce a unique system for haploid genetic screening in a model organism of regeneration. Read More

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January 2020

A post-transcriptional program of chemoresistance by AU-rich elements and TTP in quiescent leukemic cells.

Genome Biol 2020 02 10;21(1):33. Epub 2020 Feb 10.

Massachusetts General Hospital Cancer Center, Harvard Medical School, 185 Cambridge St, CPZN4202, Boston, MA, 02114, USA.

Background: Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. Read More

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February 2020

Imaging growth as a predictor of grade of malignancy and aggressiveness of IDH-mutant and 1p/19q-codeleted oligodendrogliomas in adults.

Neuro Oncol 2020 07;22(7):993-1005

Department of Neurosurgery, University Hospital Group for Psychiatry and Neurosciences (GHU)-Sainte-Anne Hospital, Paris, France.

Background: We quantified the spontaneous imaging growth rate of oligodendrogliomas. We assessed whether (i) it discriminates between World Health Organization (WHO) grade II and grade III oligodendrogliomas, and (ii) grade III oligodendrogliomas with neo-angiogenesis are associated with more fast growth rates (≥8 mm/y).

Methods: This work employed a retrospective bicentric cohort study (2010-2016) of adult patients harboring a newly diagnosed supratentorial oligodendroglioma, isocitrate dehydrogenase (IDH) mutant and 1p/19q codeleted (WHO 2016 classification), with a minimum of 2 available MRIs before any treatment (minimum 6-week interval) to measure the spontaneous tumor growth rate. Read More

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