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https://www.readbyqxmd.com/read/29150930/photoautotrophic-production-of-macular-pigment-in-a-chlamydomonas-reinhardtii-strain-generated-by-using-dna-free-crispr-cas9-rnp-mediated-mutagenesis
#1
Kwangryul Baek, Jihyeon Yu, Jooyeon Jeong, Sang Jun Sim, Sangsu Bae, EonSeon Jin
Lutein and zeaxanthin are dietary carotenoids reported to be protective against age-related macular degeneration. Recently, the green alga Chlamydomonas reinhardtii has received attention as a photosynthetic cell factory, but the potential of this alga for carotenoid production has not yet been evaluated. In this study, we selected the C. reinhardtii CC-4349 strain as the best candidate among seven laboratory strains tested for carotenoid production. A knock-out mutant of the zeaxanthin epoxidase gene induced by preassembled DNA-free CRISPR-Cas9 ribonucleoproteins in the CC-4349 strain had a significantly higher zeaxanthin content (56-fold) and productivity (47-fold) than the wild type without the reduction in lutein level...
November 18, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/29150636/a-genetic-system-for-targeted-mutations-to-disrupt-and-restore-genes-in-the-obligate-bacterium-ehrlichia-chaffeensis
#2
Ying Wang, Lanjing Wei, Huitao Liu, Chuanmin Cheng, Roman R Ganta
Obligate intracellular bacteria (obligates) belonging to Rickettsiales and Chlamydiales cause diseases in hundreds of millions of people worldwide and in many animal species. Lack of an efficient system for targeted mutagenesis in obligates remains a major impediment in understanding microbial pathogenesis. Challenges in creating targeted mutations may be attributed to essential nature of majority of the genes and intracellular replication dependence. Despite success in generating random mutations, a method that works well in creating mutations in specific genes of interest followed by complementation remains problematic for obligates and is a highly sought-after goal...
November 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29149514/isolation-and-characterization-of-high-protein-and-phycocyanin-producing-mutants-of-arthrospira-platensis
#3
Geeta G Shirnalli, Manish S Kaushik, Ajay Kumar, Gerard Abraham, Pawan K Singh
Cyanobacteria are known to exhibit their efficiency in producing high concentrations of compounds of commercial value. Arthrospira is one such cyanobacterium which is considered as important source of protein (65%) and other nutrients. In present study, chemical mutagenesis using N-methyl-Ń-Nitro-nitrosoguanidine (NTG), a proven potent mutagen for cyanobacteria was used to bring stable and desirable alteration in Arthrospira platensis ARM 730. Three morphological mutants (G-1, G-2, and SF) were selected and characterized...
November 17, 2017: Journal of Basic Microbiology
https://www.readbyqxmd.com/read/29148725/chromophore-isomer-stabilization-is-critical-to-efficient-fluorescence-in-cyan-fluorescent-proteins
#4
Guillaume Gotthard, David von Stetten, Damien Clavel, Marjolaine Noirclerc-Savoye, Antoine Royant
ECFP, the first usable Cyan Fluorescent Protein, was obtained by adapting the tyrosine-based chromophore environment in Green Fluorescent Protein to that of a tryptophan-based one. This 1(st)-generation CFP was superseded by the popular Cerulean, CyPet and SCFP3A that were engineered by rational and random mutagenesis. Yet, the latter CFPs still exhibit suboptimal properties of pH sensitivity and reversible photobleaching behavior. These flaws were serendipitously corrected in the 3(rd)-generation CFP mTurquoise and its successors without obvi-ous rationale...
November 17, 2017: Biochemistry
https://www.readbyqxmd.com/read/29146998/regulation-of-gene-expression-and-rna-editing-in-drosophila-adapting-to-divergent-microclimates
#5
Arielle L Yablonovitch, Jeremy Fu, Kexin Li, Simpla Mahato, Lin Kang, Eugenia Rashkovetsky, Abraham B Korol, Hua Tang, Pawel Michalak, Andrew C Zelhof, Eviatar Nevo, Jin Billy Li
Determining the mechanisms by which a species adapts to its environment is a key endeavor in the study of evolution. In particular, relatively little is known about how transcriptional processes are fine-tuned to adjust to different environmental conditions. Here we study Drosophila melanogaster from 'Evolution Canyon' in Israel, which consists of two opposing slopes with divergent microclimates. We identify several hundred differentially expressed genes and dozens of differentially edited sites between flies from each slope, correlate these changes with genetic differences, and use CRISPR mutagenesis to validate that an intronic SNP in prominin regulates its editing levels...
November 17, 2017: Nature Communications
https://www.readbyqxmd.com/read/29146597/structure-guided-engineering-of-the-substrate-specificity-of-a-fungal-%C3%AE-glucuronidase-toward-triterpenoid-saponins
#6
Bo Lv, Hanli Sun, Shen Huang, Xudong Feng, Tao Jiang, Chun Li
Glycoside hydrolases (GHs) have attracted special attention in research aimed at modifying natural products by partial removal of sugar moieties to manipulate their solubility and efficacy. However, these modifications are challenging to control because the low substrate specificity of most GHs often generates undesired by-products. We previously identified a GH2-type fungal β-glucuronidase from Aspergillus oryzae (PGUS) exhibiting promiscuous substrate specificity in hydrolysis of triterpenoid saponins. Here, we present the PGUS structure, representing the first structure of a fungal β-glucuronidase, and of an inactive PGUS mutant in complex with the native substrate glycyrrhetic acid 3-O-mono-β- glucuronide (GAMG)...
November 16, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29146558/role-of-his-his-interaction-in-ser-474-his-475-tyr-476-sequence-of-chondroitinase-abc-i-in-the-enzyme-activity-and-stability
#7
Monireh Maleki, Khosro Khajeh, Massoud Amanlou, Abolfazl Golestani
Despite clinical importance of chondroitinase ABC I, its application has been limited due to thermal instability as reported in the literature. There are various approaches to improve thermal stability of enzymes, among them, His-His interactions are believed generally as an effective means. In the present study and for preparing a His-His interaction, various mutations in the sequence of Ser(474)-His(475)-Tyr(476) at catalytic domain of the enzyme were performed using site directed mutagenesis method. The effect of these mutations on activity, stability and structural features of cABC I was assessed...
November 13, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/29146383/not-just-antibiotics-is-cancer-chemotherapy-driving-antimicrobial-resistance
#8
REVIEW
Lito E Papanicolas, David L Gordon, Steve L Wesselingh, Geraint B Rogers
The global spread of antibiotic-resistant pathogens threatens to increase the mortality of cancer patients significantly. We propose that chemotherapy contributes to the emergence of antibiotic-resistant bacteria within the gut and, in combination with antibiotics, drives pathogen overgrowth and translocation into the bloodstream. In our model, these processes are mediated by the effects of chemotherapy on bacterial mutagenesis and horizontal gene transfer, the disruption of commensal gut microbiology, and alterations to host physiology...
November 13, 2017: Trends in Microbiology
https://www.readbyqxmd.com/read/29145514/why-patients-with-thbd-c-1611c-a-p-cys537x-nonsense-mutation-have-high-levels-of-soluble-thrombomodulin
#9
Yohann Jourdy, Nathalie Enjolras, Sandra Le Quellec, Jean Claude Bordet, Claude Négrier, Christine Vinciguerra, Yesim Dargaud
BACKGROUND: Recently our group has described a new autosomal dominant bleeding disorder characterized by very high plasma levels of soluble thrombomodulin (TM). The THBD c.1611C>A (p.Cys537X) mutation in heterozygous state was found in the propositus. This mutation leads to the synthesis of a truncated TM which has lost the last three amino-acids of the transmembrane domain and the cytoplasmic tail. OBJECTIVE: We investigated the mechanism responsible for TM shedding in endothelial cells with THBD c...
2017: PloS One
https://www.readbyqxmd.com/read/29143824/pd-1-is-a-haploinsufficient-suppressor-of-t-cell-lymphomagenesis
#10
Tim Wartewig, Zsuzsanna Kurgyis, Selina Keppler, Konstanze Pechloff, Erik Hameister, Rupert Öllinger, Roman Maresch, Thorsten Buch, Katja Steiger, Christof Winter, Roland Rad, Jürgen Ruland
T cell non-Hodgkin lymphomas are a heterogeneous group of highly aggressive malignancies with poor clinical outcomes. T cell lymphomas originate from peripheral T cells and are frequently characterized by genetic gain-of-function variants in T cell receptor (TCR) signalling molecules. Although these oncogenic alterations are thought to drive TCR pathways to induce chronic proliferation and cell survival programmes, it remains unclear whether T cells contain tumour suppressors that can counteract these events...
November 15, 2017: Nature
https://www.readbyqxmd.com/read/29141013/indcaps-a-tool-for-designing-screening-primers-for-crispr-cas9-mutagenesis-events
#11
Charles Hodgens, Zachary L Nimchuk, Joseph J Kieber
Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles...
2017: PloS One
https://www.readbyqxmd.com/read/29140786/selection-of-an-anticalin%C3%A2-against-the-membrane-form-of-hsp70-via-bacterial-surface-display-and-its-theranostic-application-in-tumour-models
#12
Lars Friedrich, Petra Kornberger, Claudia T Mendler, Gabriele Multhoff, Markus Schwaiger, Arne Skerra
We describe the selection of Anticalins against a common tumour surface antigen, human Hsp70, using functional display on live E. coli cells as fusion with a truncated EspP autotransporter. While found intracellularly in normal cells, Hsp70 is frequently exposed in a membrane-bound state on the surface of tumour cells and, even more pronounced, in metastases or after radiochemotherapy. Employing a recombinant Hsp70 fragment comprising residues 383-548 as target, Anticalins were selected from a naive bacterial library...
November 27, 2017: Biological Chemistry
https://www.readbyqxmd.com/read/29140675/identification-of-novel-allosteric-modulators-of-glutamate-transporter-eaat2
#13
Sandhya Kortagere, Ole Valente Mortensen, Jingsheng Xia, William Lester, Yuhong Fang, Yellamelli V V Srikanth, Joseph M Salvino, Andreia Cristina Karklin Fontana
Dysfunction of excitatory amino acid transporters (EAATs) has been implicated in the pathogenesis of various neurological disorders, such as stroke, brain trauma, epilepsyand neurodegenerative diseases among others. EAAT2 is the main subtype responsible for glutamate clearance in the brain, having a key role in regulating transmission and preventing excitotoxicity. Therefore, compounds that increase the expression or activity of EAAT2 have therapeutic potential for neuroprotection. Previous studies identified molecular determinants for EAAT2 transport stimulation in a structural domain that lies at the interface of the rigid trimerization domain and the central substrate binding transport domain...
November 15, 2017: ACS Chemical Neuroscience
https://www.readbyqxmd.com/read/29140092/determinants-and-expansion-of-specificity-in-a-trichothecene-udp-glucosyltransferase-from-oryza-sativa
#14
Karl M Wetterhorn, Kaitlyn Gabardi, Herbert Michlmayr, Alexandra Malachova, Mark Busman, Susan Patricia McCormick, Franz Berthiller, Gerhard Adam, Ivan Rayment
Family 1 UDP-glycosyltransferases in plants (UGTs) primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, where this is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins on account of its ability to glycosylate DON, nivalenol and hydrolyzed T-2 toxin (HT-2)...
November 15, 2017: Biochemistry
https://www.readbyqxmd.com/read/29140084/characterization-of-a-ugt84-family-glycosyltransferase-provides-new-insights-into-substrate-binding-and-reactivity-of-galloylglucose-ester-forming-ugts
#15
Alexander E Wilson, Xiaoxue Feng, Nadia N Ono, Doron Holland, Rachel Amir, Li Tian
Galloylated plant specialized metabolites play important roles in plant-environment interactions as well as the promotion of human and animal health. The galloylation reactions are mediated by the formation of galloylglucose esters from gallic acid and UDP-glucose, catalyzed by the plant UGT84 family glycosyltransferases. To explore and exploit the structural determinants of UGT84 activities, we performed homology modeling and substrate docking of PgUGT84A23, a galloylglucose ester-forming family 84 UGT, as well as sequence comparisons of PgUGT84A23 with other functionally characterized plant UGTs...
November 15, 2017: Biochemistry
https://www.readbyqxmd.com/read/29138304/refactoring-the-genetic-code-for-increased-evolvability
#16
Gur Pines, James D Winkler, Assaf Pines, Ryan T Gill
The standard genetic code is robust to mutations during transcription and translation. Point mutations are likely to be synonymous or to preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best-performing mutant requires replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter a single nucleotide per mutation event, due to the extreme rarity of adjacent mutations...
November 14, 2017: MBio
https://www.readbyqxmd.com/read/29137983/transcriptional-regulation-of-acetoacetyl-coa-synthetase-by-sp1-in-neuroblastoma-cells
#17
Shinya Hasegawa, Masahiko Imai, Masahiro Yamasaki, Noriko Takahashi, Tetsuya Fukui
Acetoacetyl-CoA synthetase (AACS) is the enzyme responsible for cholesterol and fatty acid synthesis in the cytosol. We have previously shown that AACS has an important role in normal neuronal development and that knockdown of SREBP-2, which orchestrates cholesterol synthesis, resulted in the downregulation of AACS mRNA levels. In this study, we investigated the transcriptional mechanism of AACS in Neuro-2a, neuroblastoma cells. Luciferase assay showed that the minimal core promoter of the mouse AACS gene is located in a region with 110 bps upstream from the transcription start site...
November 11, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29137397/dna-mismatch-repair-protein-mlh1-is-required-for-tetravalent-chromium-intermediate-induced-dna-damage
#18
Timothy P Wakeman, Aimin Yang, Naresh S Dalal, Rebecca J Boohaker, Qinghua Zeng, Qiang Ding, Bo Xu
Hexavalent chromium (Cr[VI]) is associated with occupational lung cancer and poses a significant public health concern. When exposed to Cr[VI], cells rapidly internalize this compound and metabolize it to Cr[III]. Byproducts of Cr[VI] metabolism include unstable Cr[V] and Cr[IV] intermediates that are believed to be directly responsible for the genotoxicity and carcinogenicity caused by Cr[VI] exposure; however, the carcinogenic potential of the Cr intermediates and the mechanisms of Cr-induced carcinogenesis remain to be further defined...
October 13, 2017: Oncotarget
https://www.readbyqxmd.com/read/29134273/o-cdc7-kinase-where-art-thou
#19
Robert A Sclafani, Jay R Hesselberth
Although Cdc7 protein kinase is important for regulating DNA replication in all eukaryotes and is a target for cancer therapy, it has never been localized in cells. Recently, a novel molecular genomic method used by our laboratory to localize Cdc7 to regions of chromosomes. Originally, mutations in the CDC7 gene were found in the classic cdc mutant collection of Hartwell et al. (Genetics 74:267-286, 1973). The CDC7 gene was found to encode a protein kinase called DDK that has been studied for many years, establishing its precise role in the initiation of DNA replication at origins...
November 13, 2017: Current Genetics
https://www.readbyqxmd.com/read/29133876/eph-b4-regulates-adaptive-venous-remodeling-to-improve-arteriovenous-fistula-patency
#20
Clinton D Protack, Trenton R Foster, Takuya Hashimoto, Kota Yamamoto, Monica Y Lee, Jan R Kraehling, Hualong Bai, Haidi Hu, Toshihiko Isaji, Jeans M Santana, Mo Wang, William C Sessa, Alan Dardik
Low rates of arteriovenous fistula (AVF) maturation prevent optimal fistula use for hemodialysis; however, the mechanism of venous remodeling in the fistula environment is not well understood. We hypothesized that the embryonic venous determinant Eph-B4 mediates AVF maturation. In human AVF and a mouse aortocaval fistula model, Eph-B4 protein expression increased in the fistula vein; expression of the arterial determinant Ephrin-B2 also increased. Stimulation of Eph-B-mediated signaling with Ephrin-B2/Fc showed improved fistula patency with less wall thickness...
November 13, 2017: Scientific Reports
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