scholarly journals Super-Resolution Microscopy Reveals Structural Mechanisms Driving the Nanoarchitecture of a Viral Chromatin Tether

2017 ◽  
Author(s):  
Margaret J. Grant ◽  
Matthew S. Loftus ◽  
Aiola P. Stoja ◽  
Dean H. Kedes ◽  
Malcolm Mitchell Smith
Keyword(s):  
Genetic Material ◽  
Three Dimensional ◽  
Super Resolution ◽  
Nuclear Antigen ◽  
Dimensional Structure ◽  
Scaling Exponent ◽  
Three Dimensional Model ◽  
Latently Infected ◽  
Optical Reconstruction ◽  
Host Chromatin

By tethering their circular genomes (episomes) to host chromatin, DNA tumor viruses ensure retention and segregation of their genetic material during cell divisions. Despite functional genetic and crystallographic studies, there is little information addressing the three-dimensional structure of these tethers in cells, issues critical for understanding persistent infection by these viruses. Here, we have applied direct stochastic optical reconstruction microscopy (dSTORM) to establish the nanoarchitecture of tethers within cells latently infected with the oncogenic human pathogen, Kaposi's sarcoma-associated herpesvirus (KSHV). Each KSHV tether comprises a series of homodimers of the latency-associated nuclear antigen (LANA) that bind with their C-termini to the tandem array of episomal terminal repeats (TRs) and with their N-termini to host chromatin. Super-resolution imaging revealed that individual KSHV tethers possess similar overall dimensions and, in aggregate, fold to occupy the volume of a prolate ellipsoid. Using plasmids with increasing numbers of TRs, we found that tethers display polymer power-law scaling behavior with a scaling exponent characteristic of active chromatin. For plasmids containing a two-TR tether, we determined the size, separation, and relative orientation of two distinct clusters of bound LANA, each corresponding to a single TR. From these data, we have generated a three-dimensional model of the episomal half of the tether that integrates and extends previously established findings from epi-fluorescent, crystallographic, and epigenetic approaches. Our findings also validate the use of dSTORM in establishing novel structural insights into the physical basis of molecular connections linking host and pathogen genomes.

scholarly journals Superresolution microscopy reveals structural mechanisms driving the nanoarchitecture of a viral chromatin tether

2018 ◽  
Vol 115 (19) ◽  
pp. 4992-4997 ◽  
Author(s):  
Margaret J. Grant ◽  
Matthew S. Loftus ◽  
Aiola P. Stoja ◽  
Dean H. Kedes ◽  
M. Mitchell Smith
Keyword(s):  
3D Structure ◽  
Genetic Material ◽  
Nuclear Antigen ◽  
Scaling Exponent ◽  
Superresolution Microscopy ◽  
Tumor Viruses ◽  
Latently Infected ◽  
Optical Reconstruction ◽  
Structural Mechanisms ◽  
Host Chromatin

By tethering their circular genomes (episomes) to host chromatin, DNA tumor viruses ensure retention and segregation of their genetic material during cell divisions. Despite functional genetic and crystallographic studies, there is little information addressing the 3D structure of these tethers in cells, issues critical for understanding persistent infection by these viruses. Here, we have applied direct stochastic optical reconstruction microscopy (dSTORM) to establish the nanoarchitecture of tethers within cells latently infected with the oncogenic human pathogen, Kaposi’s sarcoma-associated herpesvirus (KSHV). Each KSHV tether comprises a series of homodimers of the latency-associated nuclear antigen (LANA) that bind with their C termini to the tandem array of episomal terminal repeats (TRs) and with their N termini to host chromatin. Superresolution imaging revealed that individual KSHV tethers possess similar overall dimensions and, in aggregate, fold to occupy the volume of a prolate ellipsoid. Using plasmids with increasing numbers of TRs, we found that tethers display polymer power law scaling behavior with a scaling exponent characteristic of active chromatin. For plasmids containing a two-TR tether, we determined the size, separation, and relative orientation of two distinct clusters of bound LANA, each corresponding to a single TR. From these data, we have generated a 3D model of the episomal half of the tether that integrates and extends previously established findings from epifluorescent, crystallographic, and epigenetic approaches. Our findings also validate the use of dSTORM in establishing novel structural insights into the physical basis of molecular connections linking host and pathogen genomes.

scholarly journals Mutual Correction of Faulty PCNA Subunits in Temperature-Sensitive Lethal mus209 Mutants of Drosophila melanogaster

Genetics ◽  
2000 ◽  
Vol 154 (4) ◽  
pp. 1721-1733
Author(s):  
Daryl S Henderson ◽  
Ulrich K Wiegand ◽  
David G Norman ◽  
David M Glover
Keyword(s):  
Germ Line ◽  
Position Effect ◽  
Three Dimensional ◽  
Nuclear Antigen ◽  
Dimensional Structure ◽  
Female Sterility ◽  
Temperature Sensitive ◽  
Position Effect Variegation ◽  
Dna Double Strand Breaks ◽  
Mutant Genotype

Abstract Proliferating cell nuclear antigen (PCNA) functions in DNA replication as a processivity factor for polymerases δ and ε, and in multiple DNA repair processes. We describe two temperature-sensitive lethal alleles (mus209B1 and mus2092735) of the Drosophila PCNA gene that, at temperatures permissive for growth, result in hypersensitivity to DNA-damaging agents, suppression of position-effect variegation, and female sterility in which ovaries are underdeveloped and do not produce eggs. We show by mosaic analysis that the sterility of mus209B1 is partly due to a failure of germ-line cells to proliferate. Strikingly, mus209B1 and mus2092735 interact to restore partial fertility to heteroallelic females, revealing additional roles for PCNA in ovarian development, meiotic recombination, and embryogenesis. We further show that, although mus209B1 and mus2092735 homozygotes are each defective in repair of transposase-induced DNA double-strand breaks in somatic cells, this defect is substantially reversed in the heteroallelic mutant genotype. These novel mutations map to adjacent sites on the three-dimensional structure of PCNA, which was unexpected in the context of this observed interallelic complementation. These mutations, as well as four others we describe, reveal new relationships between the structure and function of PCNA.

Simultaneous, laser-sintered, three-dimensional modelling of bony structures and soft tissue for surgical navigation of extended cholesteatoma

2009 ◽  
Vol 124 (5) ◽  
pp. 564-568 ◽  
Author(s):  
M Suzuki ◽  
Y Ogawa ◽  
T Hasegawa ◽  
S Kawaguchi ◽  
K Yukawa ◽  
...  
Keyword(s):  
Computed Tomography ◽  
Soft Tissue ◽  
Soft Tissues ◽  
Surgical Navigation ◽  
Selective Laser Sintering ◽  
Three Dimensional ◽  
Styloid Process ◽  
Dimensional Structure ◽  
Dimensional Model ◽  
Three Dimensional Model

AbstractAim:To examine the usefulness of a three-dimensional model for surgical navigation of cholesteatoma.Materials and method:A three-dimensional model was prototyped using selective laser sintering. Based on detailed computed tomography data, powder layers were laser-fused and accumulated to create a three-dimensional structure. The computed tomography threshold was adjusted to simultaneously replicate bony structures and soft tissues.Results:The cholesteatoma, major vessels and bony structures were well replicated. This laser-sintered model was used to aid surgery for recurrent cholesteatoma. The cholesteatoma, which extended from the hypotympanum through the styloid process sheath and the internal carotid artery sheath, was removed safely via a minimal skin incision.Conclusion:The laser-sintered model was useful for surgical planning and navigation in a cholesteatoma case involving complex bony structures and soft tissue.

Pseudoknot Structures in Retroviral and Bacteriophage Messenger RNA: a Family of Structurally Related RNA Pseudoknots

1997 ◽  
Vol 3 (S2) ◽  
pp. 95-96
Author(s):  
D.W. Hoffman ◽  
Z. Du ◽  
J.A. Holland ◽  
M.R. Hansen ◽  
Y. Wang ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Three Dimensional ◽  
Structural Features ◽  
Dimensional Structure ◽  
Three Dimensional Model ◽  
Single Nucleotide ◽  
Rna Pseudoknots ◽  
Gene 32 ◽  
Rna Pseudoknot ◽  
Bacteriophage T2

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the three-dimensional structure of an RNA pseudoknot with a sequence corresponding to the 5' end region of the gene 32 messenger RNA of bacteriophage T2. NMR results show that the pseudoknot contains two coaxial A-form helical stems connected by two loops. One of the loops consists of a single nucleotide, which spans the major groove of the seven base pair helical stem 2. The second loop consists of 7 nucleotides, and spans the minor groove of stem 1. A three-dimensional model of the pseudoknot that is consistent with the NMR data will be presented, and features that are likely to be important for stabilizing the pseudoknot structure will be described.A combination of NMR and phylogenetic methods were used to characterize the structural features of RNA pseudoknots that are associated with frameshift and readthrough sites within the retroviral gag-pro messenger RNA. The majority of the retroviral frameshift and readthrough sites were found to be followed by nucleotide sequences that have the potential to form pseudoknots with structures that are remarkably similar to that of the bacteriophage T2 gene 32 mRNA.

Creating a tissue-specific microdispersed matrix from a decellularized porcine liver

2020 ◽  
Vol 4 ◽  
pp. 41-50
Author(s):  
A.D. Kirillova ◽  
◽  
Yu.B. Basok ◽  
A.E. Lazhko ◽  
A. M. Grigoryev ◽  
...  
Keyword(s):  
Sodium Dodecyl ◽  
Genetic Material ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Porcine Liver ◽  
Tissue Specific ◽  
Main Components ◽  
Triton X ◽  
Injectable Form

The main problem with decellularization of liver tissue as a tissue-specific matrix/scaffold in liver bioengineered structures is the need to maximize the preservation of the original three-dimensional structure of the tissue and the main components of its extracellular matrix (ECM) while removing cells and genetic material. The attempts to use the existing protocols for the decellularization of other tissues and organs have been unsuccessful. The aim of the work is to develop a method for creation of tissue-specific microdispersed matrix from decellularized porcine liver (TMM DLp). The protocol for decellularization of porcine liver (Lp) fragments has been developed based on the complex application of chemical (sodium dodecyl sulfate and Triton X-100), biochemical (DNase I), and physical (supercritical CO2) methods for treatment the initial tissue. As a result of the found optimal conditions for decellularization of Lp with subsequent cryomicronization of DLp, the injectable form of the microdispersed tissue-specific matrix was obtained, which represents DLp microparticles with the size of 100-200 microns with the residual amount of DNA no more than 10±1.5 ng/mg (less than 1.0%), with the preservation of the microstructure and basic composition of the liver ECM. According to the assessment of biocompatible properties in vitro, TMM DLp samples meet the criteria of biological safety for cytotoxicity and hemolytic activity.

scholarly journals A quantitative super-resolution imaging toolbox for diagnosis of motile ciliopathies

2020 ◽  
Vol 12 (535) ◽  
pp. eaay0071 ◽  
Author(s):  
Zhen Liu ◽  
Quynh P. H. Nguyen ◽  
Qingxu Guan ◽  
Alexandra Albulescu ◽  
Lauren Erdman ◽  
...  
Keyword(s):  
Lung Function ◽  
A Priori ◽  
Three Dimensional ◽  
Super Resolution ◽  
Structural Defects ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Motile Cilia ◽  
Optical Reconstruction

Airway clearance of pathogens and particulates relies on motile cilia. Impaired cilia motility can lead to reduction in lung function, lung transplant, or death in some cases. More than 50 proteins regulating cilia motility are linked to primary ciliary dyskinesia (PCD), a heterogeneous, mainly recessive genetic lung disease. Accurate PCD molecular diagnosis is essential for identifying therapeutic targets and for initiating therapies that can stabilize lung function, thereby reducing socioeconomic impact of the disease. To date, PCD diagnosis has mainly relied on nonquantitative methods that have limited sensitivity or require a priori knowledge of the genes involved. Here, we developed a quantitative super-resolution microscopy workflow: (i) to increase sensitivity and throughput, (ii) to detect structural defects in PCD patients’ cells, and (iii) to quantify motility defects caused by yet to be found PCD genes. Toward these goals, we built a localization map of PCD proteins by three-dimensional structured illumination microscopy and implemented quantitative image analysis and machine learning to detect protein mislocalization, we analyzed axonemal structure by stochastic optical reconstruction microscopy, and we developed a high-throughput method for detecting motile cilia uncoordination by rotational polarity. Together, our data show that super-resolution methods are powerful tools for improving diagnosis of motile ciliopathies.

Three Dimensional Dynamic Monitoring Model of Groundwater Resources in Jiao Nan Feng He River Basin

2013 ◽  
Vol 664 ◽  
pp. 211-214
Author(s):  
Dao Lei Xie ◽  
Jiu Chuan Wei ◽  
Hui Yong Yin ◽  
Jian Bin Guo ◽  
Ming Xiang He ◽  
...  
Keyword(s):  
River Basin ◽  
Groundwater Level ◽  
Groundwater Modeling ◽  
Groundwater Resources ◽  
Three Dimensional ◽  
Dynamic Monitoring ◽  
Water Model ◽  
Dimensional Structure ◽  
Topographic Map ◽  
Three Dimensional Model

Build strata three-dimensional structure model By GMS (Groundwater Modeling System) software about the Feng He River basin and on the basis of it place the groundwater water model to better show the groundwater level, buried depth, thickness, etc., at the same time display the locations of groundwater cordon. Groundwater cordon can be cut along with the strata and water and displayed on the cutting profile. Superimpose the model and satellite topographic map, so can select a specific position cutting three-dimensional model in order to understand a specific location or area of underground water level, water thickness, etc. Through the real-time update of groundwater level data to understand the utilization of groundwater mining in the study area, ensure the sustainable use of groundwater resources and realization of three dimensional dynamic monitoring of groundwater resources in the study area.

scholarly journals Amino-acid sequence and predicted three-dimensional structure of pea seed (Pisum sativum) ferritin

1992 ◽  
Vol 288 (3) ◽  
pp. 931-939 ◽  
Author(s):  
S Lobreaux ◽  
S J Yewdall ◽  
J F Briat ◽  
P M Harrison
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Three Dimensional ◽  
Transit Peptide ◽  
Amino Acid Identity ◽  
Dimensional Structure ◽  
Three Dimensional Model ◽  
Iron Storage ◽  
Liver Ferritin ◽  
Pea Seed

The iron storage protein, ferritin, is widely distributed in the living kingdom. Here the complete cDNA and derived amino-acid sequence of pea seed ferritin are described, together with its predicted secondary structure, namely a four-helix-bundle fold similar to those of mammalian ferritins, with a fifth short helix at the C-terminus. An N-terminal extension of 71 residues contains a transit peptide (first 47 residues) responsible for plastid targetting as in other plant ferritins, and this is cleaved before assembly. The second part of the extension (24 residues) belongs to the mature subunit; it is cleaved during germination. The amino-acid sequence of pea seed ferritin is aligned with those of other ferritins (49% amino-acid identity with H-chains and 40% with L-chains of human liver ferritin in the aligned region). A three-dimensional model has been constructed by fitting the aligned sequence to the coordinates of human H-chains, with appropriate modifications. A folded conformation with an 11-residue helix is predicted for the N-terminal extension. As in mammalian ferritins, 24 subunits assemble into a hollow shell. In pea seed ferritin, its N-terminal extension is exposed on the outside surface of the shell. Within each pea subunit is a ferroxidase centre resembling those of human ferritin H-chains except for a replacement of Glu-62 by His. The channel at the 4-fold-symmetry axes defined by E-helices, is predicted to be hydrophilic in plant ferritins, whereas it is hydrophobic in mammalian ferritins.

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