scholarly journals Identification and characterization of influenza variants resistant to a viral endonuclease inhibitor

2016 ◽  
Vol 113 (13) ◽  
pp. 3669-3674 ◽  
Author(s):  
Min-Suk Song ◽  
Gyanendra Kumar ◽  
William R. Shadrick ◽  
Wei Zhou ◽  
Trushar Jeevan ◽  
...  
Keyword(s):  
Point Mutations ◽  
Random Mutagenesis ◽  
Resistant Mutant ◽  
Inhibitor Binding ◽  
New Class ◽  
Identification And Characterization ◽  
Modest Reduction

The influenza endonuclease is an essential subdomain of the viral RNA polymerase. It processes host pre-mRNAs to serve as primers for viral mRNA and is an attractive target for antiinfluenza drug discovery. Compound L-742,001 is a prototypical endonuclease inhibitor, and we found that repeated passaging of influenza virus in the presence of this drug did not lead to the development of resistant mutant strains. Reduced sensitivity to L-742,001 could only be induced by creating point mutations via a random mutagenesis strategy. These mutations mapped to the endonuclease active site where they can directly impact inhibitor binding. Engineered viruses containing the mutations showed resistance to L-742,001 both in vitro and in vivo, with only a modest reduction in fitness. Introduction of the mutations into a second virus also increased its resistance to the inhibitor. Using the isolated wild-type and mutant endonuclease domains, we used kinetics, inhibitor binding and crystallography to characterize how the two most significant mutations elicit resistance to L-742,001. These studies lay the foundation for the development of a new class of influenza therapeutics with reduced potential for the development of clinical endonuclease inhibitor-resistant influenza strains.

scholarly journals Long read sequencing reveals a novel class of structural aberrations in cancers: identification and characterization of cancerous local amplifications

2019 ◽  
Author(s):  
Yoshitaka Sakamoto ◽  
Liu Xu ◽  
Masahide Seki ◽  
Toshiyuki T. Yokoyama ◽  
Masahiro Kasahara ◽  
...  
Keyword(s):  
Point Mutations ◽  
Lung Cancers ◽  
New Class ◽  
Large Deletions ◽  
Long Read ◽  
Structural Aberrations ◽  
Molecular Etiology ◽  
Identification And Characterization

AbstractHere we report identification of a new class of local structural aberrations in lung cancers. The whole-genome sequencing of cell lines using a long read sequencer, PromethION, demonstrated that typical cancerous mutations, such as point mutations, large deletions and gene fusions can be detected also on this platform. Unexpectedly, we revealed unique structural aberrations consisting of complex combinations of local duplications, inversions and micro deletions. We further analyzed and found that these mutations also occur in vivo, even in key cancer-related genes. These mutations may elucidate the molecular etiology of patients for whom causative cancerous events and therapeutic strategies remain elusive.

scholarly journals A primary study on genes with selected mutations by in vitro passage of Chlamydia muridarum strains

2019 ◽  
Vol 77 (3) ◽  
Author(s):  
Zhou Zhou ◽  
Na Liu ◽  
Yingzi Wang ◽  
Arthur Wirekoh Emmanuel ◽  
Xiaoxing You ◽  
...  
Keyword(s):  
Single Gene ◽  
Point Mutations ◽  
Primary Study ◽  
Chlamydia Muridarum ◽  
In Vivo Experiments ◽  
High Incidence ◽  
Identification And Characterization

ABSTRACTObjectiveThis study is to investigate the functions of newly discovered genes in Chlamydia muridarum (C. muridarum) strains with single gene differences.MethodsUsing whole genome sequencing and plaque formation assays, C. muridarum parental and passaging strains were established, and the isogenic clones expressing certain genotypes were isolated. Strains with single gene differences were obtained. Based on prediction, the valuable strains with single gene differences of tc0412, tc0668 or tc0237 were subjected to the in vitro and in vivo experiments for biological characterization and virulence analysis.ResultsInsertional -472840T mutation of the tc0412 gene (T28T/B3 type) matching with the nonmutant tc0668 gene and tc0237 gene with point mutations G797659T (Q117E) might slow the growth of Chlamydia due to the lack of a plasmid. The nonmutant tc0668 in the strain might induce a high incidence of hydrosalpinx in mice, while tc0668 with a G797659T point mutation was significantly attenuated. Compared with the nonmutant tc0237, the strains containing mutant tc0237 were characterized by reduced centrifugation dependence during infection.ConclusionThe identification and characterization of these genes might contribute to the comprehensive understanding of the pathogenic mechanism of Chlamydia.

scholarly journals Identification and characterization of T reg–like cells in zebrafish

2017 ◽  
Vol 214 (12) ◽  
pp. 3519-3530 ◽  
Author(s):  
Melissa Kasheta ◽  
Corrie A. Painter ◽  
Finola E. Moore ◽  
Riadh Lobbardi ◽  
Alysia Bryll ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Live Cell Imaging ◽  
Genetic Ablation ◽  
Functional Studies ◽  
Normal Functions ◽  
Inflammatory Phenotype ◽  
Identification And Characterization

Regulatory T (T reg) cells are a specialized sublineage of T lymphocytes that suppress autoreactive T cells. Functional studies of T reg cells in vitro have defined multiple suppression mechanisms, and studies of T reg–deficient humans and mice have made clear the important role that these cells play in preventing autoimmunity. However, many questions remain about how T reg cells act in vivo. Specifically, it is not clear which suppression mechanisms are most important, where T reg cells act, and how they get there. To begin to address these issues, we sought to identify T reg cells in zebrafish, a model system that provides unparalleled advantages in live-cell imaging and high-throughput genetic analyses. Using a FOXP3 orthologue as a marker, we identified CD4-enriched, mature T lymphocytes with properties of T reg cells. Zebrafish mutant for foxp3a displayed excess T lymphocytes, splenomegaly, and a profound inflammatory phenotype that was suppressed by genetic ablation of lymphocytes. This study identifies T reg–like cells in zebrafish, providing both a model to study the normal functions of these cells in vivo and mutants to explore the consequences of their loss.

scholarly journals Characterization of Spontaneous, In Vitro-Selected, Rifampin-Resistant Mutants of Mycobacterium tuberculosisStrain H37Rv

2000 ◽  
Vol 44 (12) ◽  
pp. 3298-3301 ◽  
Author(s):  
Glenn P. Morlock ◽  
Bonnie B. Plikaytis ◽  
Jack T. Crawford
Keyword(s):  
Point Mutations ◽  
Laboratory Strain ◽  
Resistant Mutant ◽  
Small Region ◽  
Clinical Isolates ◽  
Gene Coding ◽  
Resistant Mutants ◽  
Strain H37rv

ABSTRACT Resistance to rifampin in Mycobacterium tuberculosisresults from mutations in the gene coding for the beta subunit of RNA polymerase (rpoB). At least 95% of rifampin-resistant isolates have mutations in rpoB, and the mutations are clustered in a small region. About 40 distinct point mutations and in-frame insertions and deletions in rpoB have been identified, but point mutations in two codons, those coding for Ser531 and His526, are seen in about 70% of rifampin-resistant clinical isolates, with Ser531-to-Leu (TCG-to-TGG) mutations being by far the most common. To explore this phenomenon, we isolated independent, spontaneous, rifampin-resistant mutant versions of well-characterized M. tuberculosislaboratory strain H37Rv by plating 100 separate cultures, derived from a single low-density inoculum, onto rifampin-containing medium. Rifampin-resistant mutants were obtained from 64 of these cultures. Although we anticipated that the various point mutations would occur with approximately equal frequencies, sequencing the rpoBgene from one colony per plate revealed that 39 (60.9%) were Ser531 to Leu. We conclude that, for unknown reasons, the associated rpoB mutation occurs at a substantially higher rate than other rpoB mutations. This higher mutation rate may contribute to the high percentage of this mutation seen in clinical isolates.

scholarly journals Identification and Characterization of Two Bordetella avium Gene Products Required for Hemagglutination

2010 ◽  
Vol 78 (6) ◽  
pp. 2370-2376 ◽  
Author(s):  
Louise M. Temple ◽  
David M. Miyamoto ◽  
Manju Mehta ◽  
Christian M. Capitini ◽  
Stephen Von Stetina ◽  
...  
Keyword(s):  
Whooping Cough ◽  
Bioinformatic Analysis ◽  
Tracheal Ring ◽  
Tracheal Cell ◽  
Bordetella Avium ◽  
Identification And Characterization

ABSTRACT Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.

scholarly journals A Ral GAP complex links PI 3-kinase/Akt signaling to RalA activation in insulin action

2011 ◽  
Vol 22 (1) ◽  
pp. 141-152 ◽  
Author(s):  
Xiao-Wei Chen ◽  
Dara Leto ◽  
Tingting Xiong ◽  
Genggeng Yu ◽  
Alan Cheng ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Critical Role ◽  
Inhibitory Effect ◽  
Small Gtpase ◽  
Regulatory Subunit ◽  
Hydrolysis Of ◽  
Identification And Characterization

Insulin stimulates glucose transport in muscle  and adipose tissue by translocation of glucose transporter 4 (GLUT4) to the plasma membrane. We previously reported that activation of the small GTPase RalA downstream of PI 3-kinase plays a critical role in this process by mobilizing the exocyst complex for GLUT4 vesicle targeting in adipocytes. Here we report the identification and characterization of a Ral GAP complex (RGC) that mediates the activation of RalA downstream of the PI 3-kinase/Akt pathway. The complex is composed of an RGC1 regulatory subunit and an RGC2 catalytic subunit (previously identified as AS250) that directly stimulates the guanosine triphosphate hydrolysis of RalA. Knockdown of RGC proteins leads to increased RalA activity and glucose uptake in adipocytes. Insulin inhibits the GAP complex through Akt2-catalyzed phosphorylation of RGC2 in vitro and in vivo, while activated Akt relieves the inhibitory effect of RGC proteins on RalA activity. The RGC complex thus connects PI 3-kinase/Akt activity to the transport machineries responsible for GLUT4 translocation.

scholarly journals Computational Insight into the Effect of Natural Compounds on the Destabilization of Preformed Amyloid-β(1-40) Fibrils

2018 ◽  
Author(s):  
Francesco Tavanti ◽  
Alfonso Pedone ◽  
Maria Cristina Menziani
Keyword(s):  
Therapeutic Strategy ◽  
Therapeutic Potential ◽  
Structure Stability ◽  
Insoluble Form ◽  
The Brain ◽  
Identification And Characterization ◽  
Insight Into

One of the principal hallmarks of Alzheimer’s disease (AD) is related to the aggregation of amyloid-β fibrils in an insoluble form in the brain, also known as amyloidosis. Therefore, a prominent therapeutic strategy against AD consists either in blocking the amyloid aggregation and/or destroying the already formed aggregates. Natural products have shown significant therapeutic potential as amyloid inhibitors from in vitro studies as well as in vivo animal tests. In this study, the interaction of five natural biophenols (curcumin, dopamine, (-)-Epigallocatechin-3-gallate, Quercetin, and Rosmarinic acid) with the amyloid-β(1-40) fibrils has been studied through computational simulations. The results allowed the identification and characterization of the different binding modalities of each compounds and their consequences on fibril dynamics and aggregation. It emerges that the lateral aggregation of the fibrils is strongly influenced by the intercalation of the ligands, which modulate the double-layered structure stability.

Identification and Characterization of Metabolites of Irinotecan in-vivo and in-vitro matrixes by HPLC/LC-MS

2016 ◽  
Vol 2 (3) ◽  
pp. 211-218
Author(s):  
Nidhi Srivastava ◽  
Vishal Dubey ◽  
Madhumita Sengar ◽  
Rastogi Sameer
Keyword(s):  
Method Development ◽  
Biological Fluids ◽  
Parent Compound ◽  
Enzymatic Reaction ◽  
Liver Microsomes ◽  
Metabolite Identification ◽  
Identification And Characterization

In the present study metabolite identification and characterization has done by using HPLC and LC-MS. During method development various mobile phases have tried for identification of metabolites. The matrixes selected for in- vivo study were urine because nearly all the metabolites of irinotecan were obtained in it. The extraction mixtures have selected to retain maximum amount of analyte with less effort. During experiment four extraction solvents were used in six different concentrations out of which TBME suit our method. In-vitro study done by Human Liver microsomes by using Phosphate buffer (pH 7.4) and NADPH as co-factors for initiation of enzymatic reaction. Irinotecan is a prodrug that is converted in the liver to an active metabolite, SN-38. It is eliminate in Bile and Faeces and thus its dose reduced in Hepatic Failure. Irinotecan act by inhibiting Topoisomerase-1.It is the enzyme which nicks, introduces negative supercoils and reseals the DNA strand. Conventionally, drug metabolite identification in the past has usually been based on the comparison of ultraviolet (UV) spectral data and high-performance liquid chromatography (HPLC) retention times of isolated ‘unknown’ metabolites with those of synthesised standards. Such a method of detecting and characterising drug metabolites is an uncertain, time-consuming and expensive process, as well as affording very limited structural information. Furthermore, Phase I metabolism of a drug candidate often results in only minor structural modification of the parent compound; these minor changes can make it particularly difficult to determine suitable chromatographic conditions to effect HPLC separation of metabolites. This study describes contemporary approach to identification and characterization of xenobiotic metabolites in complex biological fluids derived from drug metabolism studies.

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