scholarly journals Characterization of exogenous DNA mobility in live cells through fluctuation correlation spectroscopy

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Stephen Mieruszynski ◽  
Michelle A. Digman ◽  
Enrico Gratton ◽  
Mark R Jones
Keyword(s):  
Correlation Spectroscopy ◽  
Live Cells ◽  
Exogenous Dna ◽  
Dna Mobility

Characterization of a Motile Cellular Domain Arising During the Amoebo-Flagellate Transformation of Physarum Polycephalum

1980 ◽  
Vol 38 ◽  
pp. 552-553
Author(s):  
K.I. Pagh ◽  
M.R. Adelman
Keyword(s):  
Cell Shape ◽  
Physarum Polycephalum ◽  
Slime Mold ◽  
Live Cells ◽  
Cellular Domain

Unicellular amoebae of the slime mold Physarum polycephalum undergo marked changes in cell shape and motility during their conversion into flagellate swimming cells (l). To understand the processes underlying motile activities expressed during the amoebo-flagellate transformation, we have undertaken detailed investigations of the organization, formation and functions of subcellular structures or domains of the cell which are hypothesized to play a role in movement. One focus of our studies is on a structure, termed the “ridge” which appears as a flattened extension of the periphery along the length of transforming cells (Fig. 1). Observations of live cells using Nomarski optics reveal two types of movement in this region:propagation of undulations along the length of the ridge and formation and retraction of filopodial projections from its edge. The differing activities appear to be associated with two characteristic morphologies, illustrated in Fig. 1.

scholarly journals Microfluidic Tools for Enhanced Characterization of Therapeutic Stem Cells and Prediction of Their Potential Antimicrobial Secretome

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 750
Author(s):  
Pasquale Marrazzo ◽  
Valeria Pizzuti ◽  
Silvia Zia ◽  
Azzurra Sargenti ◽  
Daniele Gazzola ◽  
...  
Keyword(s):  
Stem Cells ◽  
Defense Mechanisms ◽  
Live Cells ◽  
Label Free ◽  
Bacterial Diseases ◽  
Prospective Application ◽  
Therapy Strategies ◽  
Stromal Stem Cells ◽  
Different Sources

Antibiotic resistance is creating enormous attention on the development of new antibiotic-free therapy strategies for bacterial diseases. Mesenchymal stromal stem cells (MSCs) are the most promising candidates in current clinical trials and included in several cell-therapy protocols. Together with the well-known immunomodulatory and regenerative potential of the MSC secretome, these cells have shown direct and indirect anti-bacterial effects. However, the low reproducibility and standardization of MSCs from different sources are the current limitations prior to the purification of cell-free secreted antimicrobial peptides and exosomes. In order to improve MSC characterization, novel label-free functional tests, evaluating the biophysical properties of the cells, will be advantageous for their cell profiling, population sorting, and quality control. We discuss the potential of emerging microfluidic technologies providing new insights into density, shape, and size of live cells, starting from heterogeneous or 3D cultured samples. The prospective application of these technologies to studying MSC populations may contribute to developing new biopharmaceutical strategies with a view to naturally overcoming bacterial defense mechanisms.

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

scholarly journals Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged μNS for Live-Cell Visualization of Viral Factories

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Luke D. Bussiere ◽  
Promisree Choudhury ◽  
Bryan Bellaire ◽  
Cathy L. Miller
Keyword(s):  
Nonstructural Protein ◽  
Critical Role ◽  
Viral Proteins ◽  
Live Cell ◽  
Host Cells ◽  
Live Cells ◽  
Virus Protein ◽  
Mammalian Orthoreovirus ◽  
Virus Proteins

ABSTRACT Within infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs), which are sites of viral transcription, translation, assembly, and replication. The MRV nonstructural protein μNS comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells, but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6-amino-acid (CCPGCC) tetracysteine (TC) tag and FlAsH-EDT2 reagent. The TC tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore, the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live-cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in-depth studies of VF dynamics and host cell interactions. IMPORTANCE MRV has historically been used as a model to study the double-stranded RNA (dsRNA) Reoviridae family, the members of which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection but remain to be fully characterized. To study VFs, researchers have focused on visualizing the nonstructural protein μNS, which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC tag into the μNS open reading frame. Characterization of each recombinant reovirus sheds light on μNS interactions with viral proteins. Moreover, utilizing the TC-labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

scholarly journals Fluorescence Correlation and Cross-Correlation Spectroscopy Unveil Cytoplasmic mRNP Composition and Dynamics

2020 ◽  
Author(s):  
Àngels Mateu-Regué ◽  
Jan Christiansen ◽  
Christian Hellriegel ◽  
Finn Cilius Nielsen
Keyword(s):  
Life Cycle ◽  
Dynamic Analysis ◽  
Cross Correlation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Macromolecular Complexes ◽  
Fluorescence Correlation ◽  
Macromolecular Composition

ABSTRACTUnderstanding the mRNA life cycle requires analysis of the dynamic macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study mRNP complexes because they readily provide information about the molecular composition, stoichiometry, heterogeneity and dynamics of the particles. We developed FCS protocols for analysis of live cells and cellular lysates, and demonstrate the feasibility of analysing common cytoplasmic mRNPs composed of core factor YBX1, IMPs (or IGF2BPs) and their interactions with other RNA binding proteins such as PABPC1, ELAVL2 (HuB), STAU1 and FMRP. FCCS corroborated previously reported RNA dependent interactions between the factors and provided an estimate of the relative overlap between the factors in the mRNPs. In this way FCS and FCCS provide a new and useful approach for the quantitative and dynamic analysis of mRNP macromolecular complexes that may complement current biochemical approaches.

scholarly journals Prevalence of Vibrio alginolyticus in Sediment Samples of River and Coastal Areas of Bangladesh

2016 ◽  
Vol 29 (1) ◽  
pp. 1-6 ◽  
Author(s):  
R Siddiqui ◽  
MM Alam ◽  
MN Naser ◽  
Y Otomo ◽  
M Yasmin ◽  
...  
Keyword(s):  
Fish Population ◽  
Vibrio Alginolyticus ◽  
Live Cells ◽  
Economic Damage ◽  
Biochemical Tests ◽  
Marine Animals ◽  
Isolation And Characterization ◽  
Vibrio Pathogens ◽  
Positive Results

Vibrio alginolyticus has been thought to be a halophilic marine bacterium that causes diarrhea, otitis media and wound infection through the consumption of raw or inappropriately cooked sea food. It is one of the main Vibrio pathogens affecting marine animals, such as marine fish, shrimp and shellfish which lead to large economic damage. Although there are reports on the presence of this organism in the coastal area of other countries, not so much work has been done on the isolation and characterization of this species in Bangladesh. The present study was, therefore, undertaken to isolate and characterize V. alginolyticus organisms isolated from the rivers (fresh water) and estuaries (brackish water) of Bangladesh. A total of 9 isolates of Vibrio species were obtained from different water bodies (three from Meghna river, two from Shangu river and four from estuary) and provisionally identified as Vibrio alginolyticus following standard biochemical tests. All these 9 strains showed same pattern of antibiotic resistance to ampicillin, streotomycin, penicillin, but sensitive to nalidixic acid. In the virulence properties test, two isolates showed positive results for toxR gene and none of the isolates showed positive results for tdh gene. Challenge experiments in Singhi fish (Heteropneustes fossi) with the live cells and the culture filtrate prepared from the V. alginolyticus showed high mortality of the fish population. All these studies suggest the presence of pathogenic V. alginolyticus strains in the river water and estuarine bodies of Bangladesh and the extracellular toxin(s) of the V. alginolyticus might be one of the causes for fish mortality.Bangladesh J Microbiol, Volume 29, Number 1, June 2012, pp 1-6

Preparation and Characterization of Coenzyme Q10-Loaded Nanostructured Lipid Carriers

2011 ◽  
Vol 694 ◽  
pp. 170-174
Author(s):  
Qiang Xia ◽  
Jia Ying Wu
Keyword(s):  
Coenzyme Q ◽  
Correlation Spectroscopy ◽  
Nanostructured Lipid Carriers ◽  
Photon Correlation ◽  
Vis Spectroscopy ◽  
Before And After ◽  
Size Growth ◽  
The Mean ◽  
Stability Results

The enhancement of stability of light sensitive CoQ10 was achieved by preparation of coenzyme Q10-loaded Nanostructured Lipid Carriers through High Pressure Homogenization (HPH). Well-dissolved lipids of CoQ10 were selected, optimized ratio of emusifiers and lipids were determined for the formulation. Obtained by photon correlation spectroscopy (PCS), the mean particle size of CoQ10-NLC was 112 ± 7 nm within 60 days after preparation. In terms of centrifugal stability, results of laser diffraction (LD) analysis eliminated the existence of aggregated particles with micrometers and showed almost no size growth before and after centrifugation. Zeta potential values were from -50 to -55 mV with pH in the range of 6.56–6.72. The concentration of CoQ10-NLC measured by UV-Vis spectroscopy was as high as 8.13 mg/mL.

scholarly journals NMR Characterization of Surface Receptor Protein Interactions in Live Cells Using Methylcellulose Hydrogels

2020 ◽  
Vol 132 (10) ◽  
pp. 3914-3918 ◽  
Author(s):  
Borja Mateos ◽  
Marco Sealey‐Cardona ◽  
Katja Balazs ◽  
Judith Konrat ◽  
Guenther Staffler ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Receptor Protein ◽  
Live Cells ◽  
Nmr Characterization ◽  
Surface Receptor
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