DAPI Method: A Novel Assay to Evaluate the In vitro Impact of Nanomaterials on Mammalian Cells

2006 ◽  
Vol 951 ◽  
Author(s):  
Pavan M. V. Raja ◽  
Jennifer Connolley ◽  
Pulickel M. Ajayan ◽  
Omkaram Nalamasu ◽  
Deanna M. Thompson
Keyword(s):  
Image Analysis ◽  
Mammalian Cells ◽  
Biological Impact ◽  
Simple Estimate ◽  
Aortic Smooth Muscle ◽  
Diphenyltetrazolium Bromide ◽  
Wide Range ◽  
The Impact ◽  
Non Destructive

ABSTRACTThe increasing importance of nanomaterial-related applications has given rise to concerns pertaining to their impact on human health. In vitro mammalian cell-based assays can provide a quick and simple estimate of the possible adverse effects of the nanomaterials. However, recent studies have questioned the efficacy of traditional assays such as the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, in evaluating cell-nanomaterial interactions, implying the need for alternate methods. We applied image analysis to enumerate the DAPI (2-[4-(Aminomethyl) phenyl]-1H-indole-6-carboximidamide, dihydrochloride) – stained cellular nuclei. Image analysis, being non-destructive, capable of automation, and applicable over a wide range of cell seeding densities, offers several advantages compared to older methods like the MTT assay and hemocytometry. Using image analysis, the impact of singlewalled carbon nanotubes (SWNT) on rat aortic smooth muscle cell (SMC) growth kinetics, were examined. Despite the carbon nanomaterial presence, the fluorescent signal from the nuclei was not noticeably impacted over the SWNT range examined (0.00-0.10 mg/ml). We anticipate that this method can also be applied to evaluate the biological impact of other nanomaterials.

scholarly journals In VivoEfficacy of Anuran Trypsin Inhibitory Peptides against Staphylococcal Skin Infection and the Impact of Peptide Cyclization

2015 ◽  
Vol 59 (4) ◽  
pp. 2113-2121 ◽  
Author(s):  
U. Malik ◽  
O. N. Silva ◽  
I. C. M. Fensterseifer ◽  
L. Y. Chan ◽  
R. J. Clark ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Cyclic Peptide ◽  
Sequence Similarity ◽  
Infection Model ◽  
Content Type ◽  
Wide Range ◽  
Inhibitory Activities ◽  
The Impact

ABSTRACTStaphylococcus aureusis a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weakin vitroinhibitory activities againstS. aureus, but several had strong antibacterial activities againstS. aureusin anin vivomurine wound infection model. pYR, an immunomodulatory peptide fromRana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg−1. Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

scholarly journals Improving Anticancer Therapy with Naringenin-Loaded Silk Fibroin Nanoparticles

Nanomaterials ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 718 ◽  
Author(s):  
Marta G. Fuster ◽  
Guzmán Carissimi ◽  
Mercedes G. Montalbán ◽  
Gloria Víllora
Keyword(s):  
Silk Fibroin ◽  
Drug Loading ◽  
Drug Release Profile ◽  
Solubility In Water ◽  
Diphenyltetrazolium Bromide ◽  
Wide Range ◽  
Silk Fibroin Nanoparticles ◽  
Ea.Hy926 Cells ◽  
Cytotoxicity Effects

Naringenin (NAR), a flavonoid present in a variety of fruits, vegetables and herbs, exhibits a wide range of pharmacological effects, including anticancer activity. Nevertheless, its application in cancer therapy is limited due to its low bioavailability at the tumour site because of its poor solubility in water and slow dissolution rate. To improve the therapeutic efficacy of NAR, emergent research is looking into using nanocarriers. Silk fibroin (SF), from the Bombyx mori silkworm, is a biocompatible and biodegradable polymer with excellent mechanical properties and an amphiphilic chemistry that make it a promising candidate as a controlled release drug system. The aim of this work is to synthesize naringenin-loaded silk fibroin nanoparticles (NAR-SFNs) by dissolving the SF in the ionic liquid 1-ethyl-3-methylimidazolium acetate, using high-power ultrasounds and rapid desolvation in methanol followed by the adsorption of NAR. The NAR-SFNs were characterized by dynamic light scattering, Fourier transform infrared spectroscopy and thermogravimetric analysis. The drug loading content and encapsulation efficiency were calculated. The drug release profile best fitted a first order equation. The cytotoxicity effects of free NAR, bare silk fibroin nanoparticles (SFNs) and NAR-SFNs were assessed on HeLa and EA.hy926 cells via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results demonstrated the higher in vitro anticancer potential of synthesized NAR-SFNs than that of free NAR in HeLa cancer cells.

scholarly journals Sensing through Non-Sensing Ocular Ion Channels

2020 ◽  
Vol 21 (18) ◽  
pp. 6925
Author(s):  
Meha Kabra ◽  
Bikash Ranjan Pattnaik
Keyword(s):  
Ion Channels ◽  
Visual Processing ◽  
Signal Transmission ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cone Dystrophy ◽  
Wide Range ◽  
The Impact

Ion channels are membrane-spanning integral proteins expressed in multiple organs, including the eye. In the eye, ion channels are involved in various physiological processes, like signal transmission and visual processing. A wide range of mutations have been reported in the corresponding genes and their interacting subunit coding genes, which contribute significantly to an array of blindness, termed ocular channelopathies. These mutations result in either a loss- or gain-of channel functions affecting the structure, assembly, trafficking, and localization of channel proteins. A dominant-negative effect is caused in a few channels formed by the assembly of several subunits that exist as homo- or heteromeric proteins. Here, we review the role of different mutations in switching a “sensing” ion channel to “non-sensing,” leading to ocular channelopathies like Leber’s congenital amaurosis 16 (LCA16), cone dystrophy, congenital stationary night blindness (CSNB), achromatopsia, bestrophinopathies, retinitis pigmentosa, etc. We also discuss the various in vitro and in vivo disease models available to investigate the impact of mutations on channel properties, to dissect the disease mechanism, and understand the pathophysiology. Innovating the potential pharmacological and therapeutic approaches and their efficient delivery to the eye for reversing a “non-sensing” channel to “sensing” would be life-changing.

scholarly journals The impact of cryopreservation on bone marrow-derived mesenchymal stem cells: a systematic review

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Soukaina Bahsoun ◽  
Karen Coopman ◽  
Elizabeth C. Akam
Keyword(s):  
Systematic Review ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cell Types ◽  
Surface Marker Expression ◽  
Wide Range ◽  
And Migration ◽  
The Impact

AbstractMesenchymal stem cells (MSCs) represent an invaluable asset for the field of cell therapy. Human Bone marrow-derived MSCs (hBM-MSCs) are one of the most commonly used cell types in clinical trials. They are currently being studied and tested for the treatment of a wide range of diseases and conditions. The future availability of MSCs therapies to the public will require a robust and reliable delivery process. Cryopreservation represents the gold standard in cell storage and transportation, but its effect on BM-MSCs is still not well established. A systematic review was conducted to evaluate the impact of cryopreservation on BM-MSCs and to attempt to uncover the reasons behind some of the controversial results reported in the literature. Forty-one in vitro studies were analysed, and their results organised according to the cell attributes they assess. It was concluded that cryopreservation does not affect BM-MSCs morphology, surface marker expression, differentiation or proliferation potential. However, mixed results exist regarding the effect on colony forming ability and the effects on viability, attachment and migration, genomic stability and paracrine function are undefined mainly due to the huge variabilities governing the cryopreservation process as a whole and to the lack of standardised assays.

scholarly journals Apotransferrin in Combination with Ciprofloxacin Slows Bacterial Replication, Prevents Resistance Amplification, and Increases Antimicrobial Regimen Effect

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Paul G. Ambrose ◽  
Brian D. VanScoy ◽  
Brian M. Luna ◽  
Jun Yan ◽  
Amber Ulhaq ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Bactericidal Effect ◽  
Bacterial Density ◽  
Infection Model ◽  
Time Curve ◽  
Content Type ◽  
Wide Range ◽  
Bacterial Replication ◽  
The Impact

ABSTRACT There has been renewed interest in combining traditional small-molecule antimicrobial agents with nontraditional therapies to potentiate antimicrobial effects. Apotransferrin, which decreases iron availability to microbes, is one such approach. We conducted a 48-h one-compartment in vitro infection model to explore the impact of apotransferrin on the bactericidal activity of ciprofloxacin. The challenge panel included four Klebsiella pneumoniae isolates with ciprofloxacin MIC values ranging from 0.08 to 32 mg/liter. Each challenge isolate was subjected to an ineffective ciprofloxacin monotherapy exposure (free-drug area under the concentration-time curve over 24 h divided by the MIC [AUC/MIC ratio] ranging from 0.19 to 96.6) with and without apotransferrin. As expected, the no-treatment and apotransferrin control arms showed unaltered prototypical logarithmic bacterial growth. We identified relationships between exposure and change in bacterial density for ciprofloxacin alone (R2 = 0.64) and ciprofloxacin in combination with apotransferrin (R2 = 0.84). Addition of apotransferrin to ciprofloxacin enabled a remarkable reduction in bacterial density across a wide range of ciprofloxacin exposures. For instance, at a ciprofloxacin AUC/MIC ratio of 20, ciprofloxacin monotherapy resulted in nearly 2 log10 CFU increase in bacterial density, while the combination of apotransferrin and ciprofloxacin resulted in 2 log10 CFU reduction in bacterial density. Furthermore, addition of apotransferrin significantly reduced the emergence of ciprofloxacin-resistant subpopulations compared to monotherapy. These data demonstrate that decreasing the rate of bacterial replication with apotransferrin in combination with antimicrobial therapy represents an opportunity to increase the magnitude of the bactericidal effect and to suppress the growth rate of drug-resistant subpopulations.

scholarly journals Plasmodium falciparum: In Vitro Cytotoxicity Testing Using MTT

1998 ◽  
Vol 3 (1) ◽  
pp. 49-53 ◽  
Author(s):  
J. Enrico Lazaro ◽  
Frederick Gay
Keyword(s):  
Plasmodium Falciparum ◽  
Culture System ◽  
Inhibitory Concentration ◽  
In Vitro Cytotoxicity ◽  
Mtt Method ◽  
Diphenyltetrazolium Bromide ◽  
Wide Range ◽  
In Vitro Culture System ◽  
Comparative Results

The microculture tetrazolium assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was used to estimate the 50% inhibitory concentration of chloroquine, quinine, artemisinin, and atovaquone using a Plasmodium falciparum in vitro culture system. The MTT assay was compared to the standard tritiated hypoxan-thine assay and to a previously described method, the 2,2′-di-p-nitrophenyl-5,5′-diphenyl-3,3′-[3,3′-dimethoxy-4,4′-diphenylenel-ditetrazolium chloride (NBT) assay. In general, the results show that the three assays generate comparative results. The results of this study suggest that the MTT method is able to give a profile of cytotoxic dose response effects over a wide range of concentrations of a drug. The method may be used in work that does not require extreme pre-cision and sensitivity, for instance, as a portable rapid screen to assay natural products for in vitro cytotoxic ac-tivity against Plasmodium falciparum.

scholarly journals In-Vitro Evaluation of Biological Activity of New Series of 1, 3, 4-Oxadiazole Derivatives Against Bap Gene Expression in Acinetobacter bumanni Biofilm

2021 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Sedigheh Akbarnezhad Ghareh Lar ◽  
Nakisa Zarrabi Ahrabi ◽  
Yasin SarveAhrabi
Keyword(s):  
Biofilm Formation ◽  
Functional Group ◽  
Biological Activities ◽  
Antimicrobial Properties ◽  
Housekeeping Gene ◽  
Bacterial Biofilm ◽  
Wide Range ◽  
Oxadiazole Derivatives ◽  
The Impact

Background: Acinetobacter bumanni is one of the most common opportunistic pathogens in health centers that is resistant to many antibiotics due to biofilm production. 1, 3, 4-oxadiazoles have a wide range of biological activities. Objectives: The aim of this research was to examine the impact of new 1, 3, 4-oxadiazole derivatives on the expression of biofilm-associated surface protein (Bap), playing an important role in promoting the biofilm formation ability of A. baumannii strains. Methods: Derivatives of 1, 3, 4-oxadiazole were synthesized through a one-step synthesis. A. baumannii strains were identified and isolated in the laboratory. The antimicrobial properties of the synthesized materials against the isolated strains were investigated. DNA, RNA, and cDNA were extracted, and the relative expression of BAP gene in A. baumannii isolates was evaluated by real-time polymerase chain reaction. Results: The compound with methoxyphenyl functional group with MIC = 62.50 mg/mL had the best inhibitory performance among all derivatives. Also, the combination of 4i reduced the expression of the Bap gene by about 24 times, but it had no effect on the expression of the 16srRNA housekeeping gene. Conclusions: 1, 3, 4-oxadiazole derivatives, especially the methoxyphenyl functional group, act as an inhibitor of bacterial biofilm formation and have the potential to be used in the pharmaceutical and biological industries.

scholarly journals Quercetin Improves the Endocrine Function of Rat Testicular Tissue Under in Vitro Conditions

2021 ◽  
Vol 70 (1-2) ◽  
pp. 1-5
Author(s):  
Filip Benko ◽  
Patrik Hrnčiar ◽  
Norbert Lukáč ◽  
Róbert Kirchner ◽  
Eva Tvrdá
Keyword(s):  
Positive Impact ◽  
Endocrine System ◽  
Testicular Tissue ◽  
Endocrine Function ◽  
Enzyme Linked Immunosorbent Assay ◽  
Beneficial Effects ◽  
Wide Range ◽  
Male Hormones ◽  
The Impact

Summary Compounds of natural origin are often used for their beneficial effects on the male endocrine system and the synthesis of steroid biomolecules in testicular tissue. One of such compounds is quercetin (QUE), which belongs to the flavonoid family and is found in a wide range of vegetables, fruits and plant products. The purpose of this study is to evaluate the impact of QUE on the endocrine function of rat testicular fragments under in vitro conditions. Testicular fragments from adult Wistar rats (n=9), cultured in the D-MEM medium with different concentrations of QUE (namely 1, 10 and 100 µmol/L) for 24 h at 37°C (5% CO2), were used in the experiment conducted. Following culture, the medium was separated and the levels of cholesterol (CHOL) and male hormones were measured. CHOL values were quantified spectrophotometrically, whereas the concentrations of androstenedione (ANDRO), dehydropeiandrosterone (DHEA) and testosterone (TEST) were quantified using the enzyme-linked immunosorbent assay (ELISA) commercial kit. The results obtained indicate that 10 µmol/L QUE significantly increased (P<0.001; P<0.05) the concentrations of all the steroid biomolecules considered (CHOL, ANDRO, DHEA and TEST) when compared to the control samples. Accordingly, our findings confirm the positive impact of QUE on the endocrine function and steroidogenesis of rat testicular tissue under in vitro conditions.

scholarly journals Cardiac optogenetics

2013 ◽  
Vol 304 (9) ◽  
pp. H1179-H1191 ◽  
Author(s):  
Emilia Entcheva
Keyword(s):  
Ion Channels ◽  
Mammalian Cells ◽  
High Specificity ◽  
Emerging Technology ◽  
Inhibitory Response ◽  
Research Activity ◽  
Spatiotemporal Resolution ◽  
And Control ◽  
The Impact

Optogenetics is an emerging technology for optical interrogation and control of biological function with high specificity and high spatiotemporal resolution. Mammalian cells and tissues can be sensitized to respond to light by a relatively simple and well-tolerated genetic modification using microbial opsins (light-gated ion channels and pumps). These can achieve fast and specific excitatory or inhibitory response, offering distinct advantages over traditional pharmacological or electrical means of perturbation. Since the first demonstrations of utility in mammalian cells (neurons) in 2005, optogenetics has spurred immense research activity and has inspired numerous applications for dissection of neural circuitry and understanding of brain function in health and disease, applications ranging from in vitro to work in behaving animals. Only recently (since 2010), the field has extended to cardiac applications with less than a dozen publications to date. In consideration of the early phase of work on cardiac optogenetics and the impact of the technique in understanding another excitable tissue, the brain, this review is largely a perspective of possibilities in the heart. It covers the basic principles of operation of light-sensitive ion channels and pumps, the available tools and ongoing efforts in optimizing them, overview of neuroscience use, as well as cardiac-specific questions of implementation and ideas for best use of this emerging technology in the heart.

scholarly journals Hydraphiles: A Rigorously Studied Class of Synthetic Channel Compounds withIn VivoActivity

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Saeedeh Negin ◽  
Bryan A. Smith ◽  
Alexandra Unger ◽  
W. Matthew Leevy ◽  
George W. Gokel
Keyword(s):  
Mammalian Cells ◽  
Ion Homeostasis ◽  
Planar Bilayers ◽  
Ion Flow ◽  
Synthetic Ion Channels ◽  
Wide Range ◽  
History Of ◽  
Polarity Sensitive ◽  
History Of Analysis

Hydraphiles are a class of synthetic ion channels that now have a twenty-year history of analysis and success. In early studies, these compounds were rigorously validated in a wide range ofin vitroassays including liposomal ion flow detected by NMR or ion-selective electrodes, as well as biophysical experiments in planar bilayers. During the past decade, biological activity was observed for these compounds including toxicity to bacteria, yeast, and mammalian cells due to stress caused by the disruption of ion homeostasis. The channel mechanism was verified in cells using membrane polarity sensitive dyes, as well as patch clamping studies. This body of work has provided a solid foundation with which hydraphiles have recently demonstrated acute biological toxicity in the muscle tissue of living mice, as measured by whole animal fluorescence imaging and histological studies. Here we review the critical structure-activity relationships in the hydraphile family of compounds and thein vitroandin celluloexperiments that have validated their channel behavior. This report culminates with a description of recently reported efforts in which these molecules have demonstrated activity in living mice.

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