scholarly journals DypFISH: Dynamic Patterned FISH to Interrogate RNA and Protein Spatial and Temporal Subcellular Distribution

2019 ◽  
Author(s):  
Anca F. Savulescu ◽  
Robyn Brackin ◽  
Emmanuel Bouilhol ◽  
Benjamin Dartigues ◽  
Jonathan H. Warrell ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Localization ◽  
Temporal Distribution ◽  
Analytical Techniques ◽  
High Reproducibility ◽  
Rna Fish ◽  
Mrna Transcripts ◽  
Definition Of ◽  
Identification And Characterization

SUMMARYAdvances in single cell RNA sequencing have allowed for the identification and characterization of cellular subtypes based on quantification of the number of transcripts in each cell. However, cells may differ not only in the number of mRNA transcripts that they exhibit, but also in their spatial and temporal distribution, intrinsic to the definition of their cellular state. Here we describe DypFISH, an approach to quantitatively investigate the spatial and temporal subcellular localization of RNA and protein, by combining micropatterning of cells with fluorescence microscopy at high resolution. We introduce a range of analytical techniques for quantitatively interrogating single molecule RNA FISH data in combination with protein immunolabeling over time. Strikingly, our results show that constraining cellular architecture reduces variation in subcellular mRNA and protein distributions, allowing the characterization of their localization and dynamics with high reproducibility. Many tissues contain cells that exist in similar constrained architectures. Thus DypFISH reveals reproducible patterns of clustering, strong correlative influences of mRNA-protein localization on MTOC orientation when they are present and interdependent dynamics globally and at specific subcellular locations which can be extended to physiological systems.

scholarly journals Identification and Characterization of DM1 Patients by a New Diagnostic Certified Assay: Neuromuscular and Cardiac Assessments

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Rea Valaperta ◽  
Valeria Sansone ◽  
Fortunata Lombardi ◽  
Chiara Verdelli ◽  
Alessio Colombo ◽  
...  
Keyword(s):  
Molecular Genetic ◽  
Clinical Manifestations ◽  
Molecular Method ◽  
High Reproducibility ◽  
Pathological Mechanism ◽  
Genetic Techniques ◽  
Identification And Characterization ◽  
Very High ◽  
Unambiguous Interpretation

The expansion of the specific trinucleotide sequence, [CTG], is the molecular pathological mechanism responsible for the clinical manifestations of DM1. Many studies have described different molecular genetic techniques to detect DM1, but as yet there is no data on the analytical performances of techniques used so far in this disease. We therefore developed and validated a molecular method, “Myotonic Dystrophy SB kit,” to better characterize our DM1 population. 113 patients were examined: 20 DM1-positive, 11 DM1/DM2-negative, and13 DM1-negative/DM2-positive, who had a previous molecular diagnosis, while 69 were new cases. This assay correctly identified 113/113 patients, and all were confirmed by different homemade assays. Comparative analysis revealed that the sensitivity and the specificity of the new kit were very high (>99%). Same results were obtained using several extraction procedures and different concentrations of DNA. The distribution of pathologic alleles showed a prevalence of the “classical” form, while of the 96 nonexpanded alleles 19 different allelic types were observed. Cardiac and neuromuscular parameters were used to clinically characterize our patients and support the new genetic analysis. Our findings suggest that this assay appears to be a very robust and reliable molecular test, showing high reproducibility and giving an unambiguous interpretation of results.

scholarly journals Cytomolecular investigations using repetitive DNA probes contribute to the identification and characterization of Characidium sp. aff. C. vidali (Teleostei: Characiformes)

2021 ◽  
Vol 19 (2) ◽  
Author(s):  
Maria Lígia Marques de Oliveira ◽  
Fabilene Gomes Paim ◽  
Érica Alves Serrano de Freitas ◽  
Claudio Oliveira ◽  
Fausto Foresti
Keyword(s):  
Histone H3 ◽  
Southeastern Brazil ◽  
Chromosomal Structure ◽  
Supernumerary Chromosomes ◽  
C Banding ◽  
Coastal Streams ◽  
Definition Of ◽  
Identification And Characterization ◽  
Telomeric Probe

Abstract Characidium sp. aff. C. vidali is a species found in coastal streams in southeastern Brazil, which has karyotypic explanatory elements as the occurrence of microstructural variations, keeping the chromosomal macrostructure of the genus. The objective of this study was to apply cytomolecular tools in the chromosomes of Characidium sp. aff. C. vidali to identify characteristics in their karyotype contributing to cytogenetic definition of this species, adding information about the evolution of the chromosomal structure of the group. The species showed 2n = 50 chromosomes and from 1 to 4 additional B microchromosomes. FISH technique showed histone H3 and H4 genes in the short arm of pair 10, and microsatellites (CA)15, (CG)15, (GA)15 and (TTA)10 clustered in the subtelomeric portions of all A chromosomes, with total accumulation by supernumerary. The telomeric probe marked terminal regions of all chromosomes, in addition to the interstitial portion of four pairs, called ITS sites, with these markings being duplicated in two pairs, hence the double-ITS classification. C-banding revealed that supernumerary chromosomes are completely heterochromatic, that ITS sites are C-banding positive, but double-ITS sites are C-banding negative. So, throughout the evolution to Characidium, genomic events are occurring and restructuring chromosomes in populations.

scholarly journals Identification and characterization of bisbenzimide compounds that inhibit human cytomegalovirus replication

2021 ◽  
Vol 102 (12) ◽  
Author(s):  
Nicole Falci Finardi ◽  
HyeongJun Kim ◽  
Lee Z. Hernandez ◽  
Matthew R. G. Russell ◽  
Catherine M-K Ho ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Single Molecule ◽  
Binding Sites ◽  
Bioinformatics Analysis ◽  
Inhibitory Effect ◽  
Biochemical Assays ◽  
Hcmv Replication ◽  
Novel Targets ◽  
Identification And Characterization

The shortcomings of current anti-human cytomegalovirus (HCMV) drugs has stimulated a search for anti-HCMV compounds with novel targets. We screened collections of bioactive compounds and identified a range of compounds with the potential to inhibit HCMV replication. Of these compounds, we selected bisbenzimide compound RO-90-7501 for further study. We generated analogues of RO-90-7501 and found that one compound, MRT00210423, had increased anti-HCMV activity compared to RO-90-7501. Using a combination of compound analogues, microscopy and biochemical assays we found RO-90-7501 and MRT00210423 interacted with DNA. In single molecule microscopy experiments we found RO-90-7501, but not MRT00210423, was able to compact DNA, suggesting that compaction of DNA was non-obligatory for anti-HCMV effects. Using bioinformatics analysis, we found that there were many putative bisbenzimide binding sites in the HCMV DNA genome. However, using western blotting, quantitative PCR and electron microscopy, we found that at a concentration able to inhibit HCMV replication our compounds had little or no effect on production of certain HCMV proteins or DNA synthesis, but did have a notable inhibitory effect on HCMV capsid production. We reasoned that these effects may have involved binding of our compounds to the HCMV genome and/or host cell chromatin. Therefore, our data expand our understanding of compounds with anti-HCMV activity and suggest targeting of DNA with bisbenzimide compounds may be a useful anti-HCMV strategy.

scholarly journals The cellular niche for intestinal stem cells: a team effort

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Guoli Zhu ◽  
Jiulong Hu ◽  
Rongwen Xi
Keyword(s):  
Stem Cells ◽  
Paneth Cells ◽  
Intestinal Stem Cells ◽  
Self Renewal ◽  
Epithelial Compartment ◽  
Definition Of ◽  
Cellular Components ◽  
Identification And Characterization ◽  
Mammalian Intestine

AbstractThe rapidly self-renewing epithelium in the mammalian intestine is maintained by multipotent intestinal stem cells (ISCs) located at the bottom of the intestinal crypt that are interspersed with Paneth cells in the small intestine and Paneth-like cells in the colon. The ISC compartment is also closely associated with a sub-epithelial compartment that contains multiple types of mesenchymal stromal cells. With the advances in single cell and gene editing technologies, rapid progress has been made for the identification and characterization of the cellular components of the niche microenvironment that is essential for self-renewal and differentiation of ISCs. It has become increasingly clear that a heterogeneous population of mesenchymal cells as well as the Paneth cells collectively provide multiple secreted niche signals to promote ISC self-renewal. Here we review and summarize recent advances in the regulation of ISCs with a main focus on the definition of niche cells that sustain ISCs.

Multi-Via Electromigration Test Structures for Identification and Characterization of Different Failure Mechanisms

2005 ◽  
Vol 863 ◽  
Author(s):  
Z.S. Choi ◽  
C. W. Chang ◽  
J. H. Lee ◽  
C. L. Gan ◽  
C. V. Thompson ◽  
...  
Keyword(s):  
Current Density ◽  
Extreme Values ◽  
Electron Flow ◽  
Failure Mechanisms ◽  
Test Conditions ◽  
Zero Current ◽  
Definition Of ◽  
Fixed Structure ◽  
Identification And Characterization

AbstractExperiments on three-terminal ‘dotted-I’ test structures (copper metal lines with vias at both ends and an additional via at the center) show that the mortality of a single segment not only depends on the values of its current density and length, but also on the stress conditions in the linked segment. The current density in one 25μm long segment was fixed at 0.5MA/cm2, with electron flow toward the central via. In the othersegment, the current magnitude and sign were varied for different test populations, with the current varied from 2.5MA/cm2 to -2.5MA/cm2 with intermediate values including zero. For all cases, some test structures survived for the full 780 hours of testing and some did not. The percent of the lines that failed increased monotonically with an effective jL product defined as the maximum of the sum of the jL products from all paths through the structure. However, some lines with smaller than expected effective jL products failed, and some lines with relatively large effective jL products did not. Simulations of electromigration and electromigration-induced failures for all test conditions have been carried out. We find that test conditions leading to extreme values of the effective jL product probe different failure mechanisms than those associated with intermediate effective jL products. It is also shown that the definition of the effective jL product must be modified to account for zero current (inactive) segments that act as reservoirs or sinks. Multi-via test structures in general, and dotted-I test structures specifically, are shown to be versatile tools for identification and characterization of different failure mechanisms and length effects through the use of different test conditions with a single fixed structure.

The Dynamics of a Molecular Plug Docked onto a Solid-State Nanopore

2018 ◽  
Author(s):  
Xin Shi ◽  
Qiao Li ◽  
Rui Gao ◽  
Wei Si ◽  
Shao-Chuang Liu ◽  
...  
Keyword(s):  
Solid State ◽  
Single Molecule ◽  
Conformational Changes ◽  
Ionic Current ◽  
Observation Time ◽  
Random Coil ◽  
Dna Complexes ◽  
Dna Complex ◽  
Identification And Characterization

<a></a><a>Docking of a protein-DNA complex onto a nanopore can provide ample observation time for a detailed inspection of the complex, enabling collection of biophysical data for detection, identification, and characterization of the biomolecules. While docking of a protein-DNA complex onto a biological nanopore has enabled analytic applications of nanopores including DNA sequencing, the application of the same principle to solid-state nanopores is tempered by poor understanding of the docking process. Here, we elucidate the behaviour of individual protein-DNA complexes docked onto a solid-state nanopore by monitoring the nanopore ionic current. </a><a>Repeat docking of monovalent streptavidin-DNA complexes is found to produce ionic current blockades that fluctuate between discrete levels within the same current blockade. </a>We elucidate the roles of the protein plug and the DNA tether in the docking process, finding the docking configurations to determine the multitude of the current blockade levels whereas the frequency of the current level switching to be determined by the interactions between the molecules and the solid-state membrane. Finally, we prove the feasibility of using the nanopore docking principle for single molecule sensing using solid-state nanopores by detecting conformational changes of a tethered DNA molecule from a random coil to an i-motif states.

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