Development of Neutral Red as a pH/pCO2 Luminescent Sensor for Biological Systems

Neutral Red (NR), a eurhodin dye, is often used for staining living cells, but we demonstrated for the first time that NR can also serve as a CO2 sensor, because of NR’s unique optical properties, which change with dissolved carbon dioxide (dCO2) concentrations. In the present study, optical sensitivity of NR was quantified as a function of changes in absorption and emission spectra to dCO2 in a pH 7.3 buffer medium at eight dCO2 concentrations. NR exhibited a response time of two minutes for equilibration in pure N2 to 100% CO2 with an ~200% percent change (%∆) in emission intensity and >400%∆ in absorbance, with full reversibility. Important to its application to biological systems, NR exhibited zero sensitivity to dissolved oxygen, which has routinely caused interference for CO2 measurements. NR exhibited pH sensitive emission and excitation energies with dual excitation wavelengths at 455 nm and 540 nm, and a single emission at 640 nm. The CO2 sensing properties of NR were benchmarked by a comparison to pyranine (8-hydroxypyrene-1, 3,6-trisulfonic acid trisodium salt) (HPTS). Future studies will evaluate the feasibility of NR as an intracellular in vivo pCO2 sensor in aquatic organisms critically impacted by increasing global CO2 levels.

Download Full-text

Metallic Elements (Cu, Zn, Ni and Mn) Toxicity Effects Determination on a Fresh Water Fish Cyprinus Carpio (Common Carp) Laboratory Acclimatized

Revista de Chimie ◽

10.37358/rc.17.8.5750 ◽

2017 ◽

Vol 68 (8) ◽

pp. 1711-1715

Author(s):

Stefania Gheorghe ◽

Gabriela Geanina Vasile ◽

Cristina Gligor ◽

Irina Eugenia Lucaciu ◽

Mihai Nita Lazar

Keyword(s):

Cyprinus Carpio ◽

Aquatic Organisms ◽

Control Fish ◽

Toxicity Tests ◽

Fresh Water Fish ◽

Metallic Elements ◽

Mn Toxicity ◽

Vitro Effect

Metallic elements copper (Cu), zinc (Zn), nickel (Ni) and manganese (Mn) are some of the most commonly found in water and sediment samples collected from the Danube - Danube Delta. These elements are important as essential micronutrients, being normally present at low concentrations in biological organisms, but in high concentrations they become toxic with immediate and delayed effects. The role of this metals is still controversial, that�s why bioconcentration potential is so important. In this non-clinical study, we tested in vitro effect of heavy metals on carp, Cyprinus carpio, reproducing in vivo presence of Cu, Zn, Ni and Mn in the Romanian�s surface water. The toxicity tests were performed according to OECD 203 by detecting the average (50%) lethal concentration - LC50 on aquatic organisms (freshwater fish) at 96h. The results pointed out that, copper value for LC 50 at 96h was estimated as 3.4 mg/L (concentrations tested in the range of 0.1 - 4.75 mg/L). Zinc value for LC 50 at 96h was estimated as 20.8 mg/L (concentrations tested in the range of 0.028 � 29.6 mg/L). Nickel value for LC 50 at 96h was estimated as 40.1 mg/L (concentrations tested in the range of 0.008 - 84.5 mg/L). For manganese the mortality effects has recorded at LC 50 at 96h at estimated value higher than 53 mg/L (concentrations tested in the range of 0.04 - 53.9 mg/L). The accuracy of the testing metals concentration was insured by the screening of the dilution water, as well as food and control fish, acclimated in laboratory conditions.

Download Full-text

Development of Synchrotron Footprinting at NSLS and NSLS-II

Protein and Peptide Letters ◽

10.2174/0929866526666181128125125 ◽

2019 ◽

Vol 26 (1) ◽

pp. 55-60 ◽

Cited By ~ 1

Author(s):

Jen Bohon

Keyword(s):

Solvent Accessibility ◽

Biological Systems ◽

In Vivo Studies ◽

Aqueous Samples ◽

Biological Mechanisms ◽

Macromolecular Assembly ◽

Structure And Dynamics ◽

Synchrotron Light ◽

Ideal Technique

Background: First developed in the 1990’s at the National Synchrotron Light Source, xray synchrotron footprinting is an ideal technique for the analysis of solution-state structure and dynamics of macromolecules. Hydroxyl radicals generated in aqueous samples by intense x-ray beams serve as fine probes of solvent accessibility, rapidly and irreversibly reacting with solvent exposed residues to provide a “snapshot” of the sample state at the time of exposure. Over the last few decades, improvements in instrumentation to expand the technology have continuously pushed the boundaries of biological systems that can be studied using the technique. Conclusion: Dedicated synchrotron beamlines provide important resources for examining fundamental biological mechanisms of folding, ligand binding, catalysis, transcription, translation, and macromolecular assembly. The legacy of synchrotron footprinting at NSLS has led to significant improvement in our understanding of many biological systems, from identifying key structural components in enzymes and transporters to in vivo studies of ribosome assembly. This work continues at the XFP (17-BM) beamline at NSLS-II and facilities at ALS, which are currently accepting proposals for use.

Download Full-text

A Study on UV Filter Chemicals from Annex VII of European Union Directive 76/768/EEC, in the In Vitro 3T3 NRU Phototoxicity Test

Alternatives to Laboratory Animals ◽

10.1177/026119299802600511 ◽

1998 ◽

Vol 26 (5) ◽

pp. 679-708 ◽

Cited By ~ 5

Author(s):

Horst Spielmann ◽

Michael Balls ◽

Jack Dupuis ◽

Wolfgang J. W. Pape ◽

Odile de Silva ◽

...

Keyword(s):

Neutral Red ◽

Optimum Concentration ◽

Alternative Methods ◽

Uv Filter ◽

Eu Directive ◽

Optimum Concentration Range ◽

The Impact ◽

Positive Results

In 1996, the Scientific Committee on Cosmetology of DGXXIV of the European Commission asked the European Centre for the Validation of Alternative Methods to test eight UV filter chemicals from the 1995 edition of Annex VII of Directive 76/768/EEC in a blind trial in the in vitro 3T3 cell neutral red uptake phototoxicity (3T3 NRU PT) test, which had been scientifically validated between 1992 and 1996. Since all the UV filter chemicals on the positive list of EU Directive 76/768/EEC have been shown not to be phototoxic in vivo in humans under use conditions, only negative effects would be expected in the 3T3 NRU PT test. To balance the number of positive and negative chemicals, ten phototoxic and ten non-phototoxic chemicals were tested under blind conditions in four laboratories. Moreover, to assess the optimum concentration range for testing, information was provided on appropriate solvents and on the solubility of the coded chemicals. In this study, the phototoxic potential of test chemicals was evaluated in a prediction model in which either the Photoirritation Factor (PIF) or the Mean Photo Effect (MPE) were determined. The results obtained with both PIF and MPE were highly reproducible in the four laboratories, and the correlation between in vitro and in vivo data was almost perfect. All the phototoxic test chemicals provided a positive result at concentrations of 1μg/ml, while nine of the ten non-phototoxic chemicals gave clear negative results, even at the highest test concentrations. One of the UV filter chemicals gave positive results in three of the four laboratories only at concentrations greater than 100μg/ml; the other laboratory correctly identified all 20 of the test chemicals. An analysis of the impact that exposure concentrations had on the performance of the test revealed that the optimum concentration range in the 3T3 NRU PT test for determining the phototoxic potential of chemicals is between 0.1μg/ml and 10μg/ml, and that false positive results can be obtained at concentrations greater than 100μg/ml. Therefore, the positive results obtained with some of the UV filter chemicals only at concentrations greater than 100μg/ml do not indicate a phototoxic potential in vivo. When this information was taken into account during calculation of the overall predictivity of the 3T3 NRU PT test in the present study, an almost perfect correlation of in vitro versus in vivo results was obtained (between 95% and 100%), when either PIF or MPE were used to predict the phototoxic potential. The management team and participants therefore conclude that the 3T3 NRU PT test is a valid test for correctly assessing the phototoxic potential of UV filter chemicals, if the defined concentration limits are taken into account.

Download Full-text

Multi-Modal Multi-Spectral Intravital Microscopic Imaging of Signaling Dynamics in Real-Time during Tumor–Immune Interactions

Cells ◽

10.3390/cells10030499 ◽

2021 ◽

Vol 10 (3) ◽

pp. 499

Author(s):

Tracy W. Liu ◽

Seth T. Gammon ◽

David Piwnica-Worms

Keyword(s):

Molecular Imaging ◽

Single Cell ◽

Real Time ◽

Bioluminescence Imaging ◽

Emission Spectra ◽

Ccd Camera ◽

Microscopic Imaging ◽

Signaling Dynamics ◽

Window Chamber

Intravital microscopic imaging (IVM) allows for the study of interactions between immune cells and tumor cells in a dynamic, physiologically relevant system in vivo. Current IVM strategies primarily use fluorescence imaging; however, with the advances in bioluminescence imaging and the development of new bioluminescent reporters with expanded emission spectra, the applications for bioluminescence are extending to single cell imaging. Herein, we describe a molecular imaging window chamber platform that uniquely combines both bioluminescent and fluorescent genetically encoded reporters, as well as exogenous reporters, providing a powerful multi-plex strategy to study molecular and cellular processes in real-time in intact living systems at single cell resolution all in one system. We demonstrate that our molecular imaging window chamber platform is capable of imaging signaling dynamics in real-time at cellular resolution during tumor progression. Importantly, we expand the utility of IVM by modifying an off-the-shelf commercial system with the addition of bioluminescence imaging achieved by the addition of a CCD camera and demonstrate high quality imaging within the reaches of any biology laboratory.

Download Full-text

Lowest aqueous picomolar fluoride ions and in vivo aluminum toxicity detection by an aluminum(iii) binding chemosensor

Dalton Transactions ◽

10.1039/d0dt03901b ◽

2021 ◽

Author(s):

Monojit Das ◽

Debdeep Maity ◽

Tusar Kanta Acharya ◽

Sudip Sau ◽

Chandan Giri ◽

...

Keyword(s):

Detection Limit ◽

Aluminum Toxicity ◽

Biological Systems ◽

Water Soluble ◽

Fluoride Ions ◽

Toxicity Effect

A water-soluble PET-based chemosensor is developed which can detect Al(iii) and F− ions up to nano- and picomolar (lowest detection so far) detection limit, respectively, also utilized to establish aluminum-toxicity effect in biological systems.

Download Full-text

Comparison of in vivo ocular irritancy testing to lens optical sensitivity in culture

Experimental Eye Research ◽

10.1016/0014-4835(92)90665-f ◽

1992 ◽

Vol 55 ◽

pp. 129

Author(s):

J.G. Sivak ◽

D.D. Stuart ◽

K.L. Herbert ◽

J.A. Van Oostrom

Keyword(s):

Optical Sensitivity

Download Full-text

Enhanced xenobiotic transporter expression in normal teleost hepatocytes: response to environmental and chemotherapeutic toxins

Journal of Experimental Biology ◽

10.1242/jeb.204.2.217 ◽

2001 ◽

Vol 204 (2) ◽

pp. 217-227

Author(s):

J.A. Albertus ◽

R.O. Laine

Keyword(s):

Mammalian Cells ◽

Cellular Localization ◽

Fundulus Heteroclitus ◽

Environmental Pollutants ◽

Human Tumor ◽

Aquatic Organisms ◽

In Vivo Studies ◽

Multixenobiotic Resistance

Many aquatic organisms are resistant to environmental pollutants, probably because their inherent multi-drug-resistant protein extrusion pump (pgp) can be co-opted to handle man-made pollutants. This mechanism of multixenobiotic resistance is similar to the mechanism of multidrug resistance exhibited in chemotherapy-resistant human tumor cells. In the present study, a variety of techniques were used to characterize this toxin defense system in killifish (Fundulus heteroclitus) hepatocytes. The cellular localization and activity of the putative drug efflux system were evaluated. In addition, in vitro and in vivo studies were used to examine the range of expression of this putative drug transporter in the presence of environmental and chemotherapeutic toxins. The broad range of pgp expression generally observed in transformed mammalian cells was found in normal cells of our teleost model. Our findings suggest that the expression of the pgp gene in the killifish could be an excellent indicator of toxin levels or stressors in the environment.

Download Full-text

Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo

Journal of Biomedical Optics ◽

10.1117/1.2032388 ◽

2005 ◽

Vol 10 (4) ◽

pp. 041210 ◽

Cited By ~ 207

Author(s):

Hui Zhao ◽

Timothy C. Doyle ◽

Olivier Coquoz ◽

Flora Kalish ◽

Bradley W. Rice ◽

...

Keyword(s):

Emission Spectra ◽

Mammalian Tissue ◽

Bioluminescent Reporters

Download Full-text

Biocompatibility Evaluation of Electrospun Scaffolds of Poly (L-Lactide) with Pure and Grafted Hydroxyapatite

Journal of the Mexican Chemical Society ◽

10.29356/jmcs.v58i4.53 ◽

2017 ◽

Vol 58 (4) ◽

Author(s):

José Manuel Cornejo-Bravo ◽

Luis Jesús Villarreal-Gómez ◽

Ricardo Vera-Graziano ◽

María Raquel Vega-Ríos ◽

José Luis Pineda-Camacho ◽

...

Keyword(s):

Bacterial Adhesion ◽

Neutral Red ◽

C2c12 Cells ◽

Surrounding Tissue ◽

Neutral Red Assay ◽

Electrospun Scaffolds ◽

Muscle Tissues ◽

Hematoxylin Eosin

The objective of this work was to evaluate the biocompatibility of scaffolds of poly(L-lactide) with pure and grafted hydroxyapatite, at various concentrations of reinforcement. The biocompatibility tests were carried out in vivo in Wistar rats by implanting the material into the subcutaneous and muscle tissues from 1 to 14 weeks and evaluating the surrounding tissue stained with hematoxylin-eosin. For in vitro assays, MTT and neutral red assay were used to evaluate any cytotoxicity in Mioblast Muscle C2C12 Cells (ATCC® CRL-1772™) and Bovine Coronary Artery Endothelial Cells (BCAEC); Escherichia coli and Staphylococcus aureus were used to evaluate bacterial adhesion. All variants of scaffolds provoked a mild inflammatory response, without showing necrosis. No evidence of cytotoxicity was presented in cell viability tests and good bacterial cell adhesion was visualized for all of the materials studied.

Download Full-text

Supervised Learning Model Predicts Protein Adsorption to Nanotubes

10.1101/2021.06.19.449132 ◽

2021 ◽

Author(s):

Rebecca L Pinals ◽

Nicholas Ouassil ◽

Jackson Travis Del Bonis-O'Donnell ◽

Jeffrey W Wang ◽

Markita P Landry

Keyword(s):

Amino Acids ◽

Protein Adsorption ◽

Biological Systems ◽

Protein Corona ◽

Engineered Nanoparticles ◽

Mass Spectrometry Data ◽

In Vitro Synthesis ◽

Intractable Problem

Engineered nanoparticles are advantageous for numerous biotechnology applications, including biomolecular sensing and delivery. However, testing the compatibility and function of nanotechnologies in biological systems requires a heuristic approach, where unpredictable biofouling often prevents effective implementation. Such biofouling is the result of spontaneous protein adsorption to the nanoparticle surface, forming the "protein corona" and altering the physicochemical properties, and thus intended function, of the nanotechnology. To better apply engineered nanoparticles in biological systems, herein, we develop a random forest classifier (RFC) trained with proteomic mass spectrometry data that identifies which proteins adsorb to nanoparticles. We model proteins that populate the corona of a single-walled carbon nanotube (SWCNT)-based optical nanosensor. We optimize the classifier and characterize the classifier performance against other models. To evaluate the predictive power of our model, we then apply the classifier to rapidly identify and experimentally validate proteins with high binding affinity to SWCNTs. Using protein properties based solely on amino acid sequence, we further determine protein features associated with increased likelihood of SWCNT binding: proteins with high content of solvent-exposed glycine residues and non-secondary structure-associated amino acids. Furthermore, proteins with high leucine residue content and beta-sheet-associated amino acids are less likely to form the SWCNT protein corona. The classifier presented herein provides an important tool to undertake the otherwise intractable problem of predicting protein-nanoparticle interactions, which is needed for more rapid and effective translation of nanobiotechnologies from in vitro synthesis to in vivo use.

Download Full-text