scholarly journals The Optimized Quantum Dots Mediated Thermometry Reveals the Efficiency of Myosin Extracted from Muscle Mini Bundles

2021 ◽  
Author(s):  
Meishan Li ◽  
Lucia Coppo ◽  
Bhanu P. Jena ◽  
Lars Larsson
Keyword(s):  
Quantum Dots ◽  
Heat Release ◽  
Fluorescence Intensity ◽  
Time Course ◽  
Enzymatic Reaction ◽  
Minimal Amount ◽  
Lower Efficiency ◽  
Fluorescent Intensity ◽  
Fast Myosin

Abstract Background: The quantum dots (QD) has been investigated as thermometrical sensor in biological microenvironment and applied to measure the muscle efficiency with underlying mechanisms, i.e., a reduction in fluorescent intensity of QD reflects an increase in temperature caused by heat release during ATP hydrolysis, denoting the efficiency of the motor protein myosin. The aim of this study is to optimize the QD mediated thermometry for measuring the efficiency of freshly extracted myosin from muscle mini bundles rather than pre-purified myosin and test this approach in preparations with different myosin isoform.Methods: The protocol of myosin extraction used in the single muscle fiber in vitro motility assay was modified slightly for extracting myosin from the muscle mini bundles. Moreover, the quantitation of extracted myosin was calculated from the total extracted protein since the ratio of myosin to total protein was constant, performing through spectrophotometric measurement of UV absorbance at 280 nm. The change in fluorescence intensity of QD thermometry measurement of myosin ATPase enzymatic reaction was plotted over time, and the slope of the linear negative regression between time course and relatively decreased fluorescence intensity was used to reflect the efficiency of extracted myosin. Results: The optimized QD mediated thermometry is established for evaluating the efficiency of myosin extracted from muscle mini bundles. Moreover, myosin isoform specific differences in the myosin efficiency were observed in comparison between slow myosin and fast myosin, i.e., the low myosin has lower efficiency than fast myosin, evidenced by a higher heat release.Conclusions: The optimized QD mediated thermometric measure of myosin efficiency in muscle mini bundles provides a nanoscale approach to evaluate myosin function based on a minimal amount of muscle, which is essentially required in the muscle research.

Quantum Dots CdSe/ZnS-Loaded Poly(D,L-Lactide-Co-Glycolide) Nanoparticles: Physicochemical Characterization and Application

2006 ◽  
Vol 505-507 ◽  
pp. 667-672 ◽  
Author(s):  
Chih Hui Yang ◽  
Kuo Chin Lin ◽  
Yu Huai Chang ◽  
Yu Cheng Lin
Keyword(s):  
Quantum Dots ◽  
Fluorescence Intensity ◽  
Fluorescence Spectra ◽  
Uv Light ◽  
Physicochemical Characterization ◽  
Plga Nanoparticles ◽  
Optimum Concentration ◽  
Cellular Internalization ◽  
Optimum Concentration Range

This paper described and characterized the quantum dots (QDs) with/without the polymeric PLGA applied in MC3T3E-1 delivery. Neat QDs were treated with various solvents, temperatures, exposure time and concentration to evaluate their stability and efficacy. We found that the intensity degree of fluorescence spectra (QDs) in different solvents follows the order: ether > THF > acetone > chloroform > methanol. Importantly, the QDs become inactive after 8-hr dissolution in the solvents of ether, THF or chloroform. According to this result, acetone and methanol are ideal solvents for QDs. The optimum concentration range of QDs in acetone is 5 to 10 mg/mL. We found that no obvious difference of fluorescence intensity was detected in QDs stored respectively at 4 °C, 24 °C and 44 °C (8-hour). When QDs were exposed to UV light (312 nm) for 2 hr, serious decay of fluorescence intensity was observed. In order to extend the application of QDs in medical areas, we encapsulated them in individual biocompatible poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles for in-vitro imaging of endocytosis in MC3T3E-1 cells. We demonstrated that the polymeric PLGA have the ability to permeate the cells for cellular internalization; the endocytotic activity could be enhanced by the polymeric QDs-encapsulated PLGA.

An in vitro study of Hoechst 33342 redistribution and its effects on cell viability

2005 ◽  
Vol 24 (11) ◽  
pp. 573-580 ◽  
Author(s):  
N Mohorko ◽  
N Kregar-Velikonja ◽  
G Repovs ◽  
M Gorensek ◽  
M Bresjanac
Keyword(s):  
Cell Death ◽  
Cell Transplantation ◽  
Fluorescence Intensity ◽  
Time Course ◽  
In Vitro Study ◽  
Host Cells ◽  
Hoechst 33342 ◽  
Overnight Incubation ◽  
Donor Cells

Although Hoechst 33342 (H342) is frequently used to label donor cells in cell transplantation research, it has been noted that it might secondarily label the host cells. Furthermore, its potential toxicity leading to cell death has been described. We studied the time course of H342 redistribution from the primary labeled rat bone marrow stromal cells (rBMSC) into the non-labeled rBMSC population over 7 days in culture; we evaluated the nuclear H342 fluorescence intensity as a possible criterion for distinguishing the primary from the secondary labeled cells, and determined the viability of rBMSC after an overnight incubation in 1 mg/mL of H342. H342 labeled / 50% of the initially non-labeled cells within the first 6 hours and almost 90% within a week.Nuclear fluorescence intensity was a reliable criterion for distinguishing primary and secondary labeled cells within the first 24 hours, but less so at later time points. The percentage of either apoptotic or necrotic cells did not rise acutely after the overnight incubation in 1 mg/mL of H342. Although a 12-hour incubation of rBMSC in 1 mg/mL of H342 did not cause acute cell death, H342 rapidly and extensively redistributed into non-labeled cells, which makes H342 a relatively unsuitable marker for cell transplantation research.

Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

1991 ◽  
Vol 66 (05) ◽  
pp. 609-613 ◽  
Author(s):  
I R MacGregor ◽  
J M Ferguson ◽  
L F McLaughlin ◽  
T Burnouf ◽  
C V Prowse
Keyword(s):  
High Purity ◽  
Time Course ◽  
Prothrombin Complex Concentrate ◽  
Degradation Products ◽  
Canine Model ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

The Effect of Heparin vs. Citrate on the Interaction of Platelets with Vascular Graft Materials

1985 ◽  
Vol 54 (04) ◽  
pp. 842-848 ◽  
Author(s):  
Kandice Kottke-Marchant ◽  
James M Anderson ◽  
Albert Rabinovitch ◽  
Richard A Huskey ◽  
Roger Herzig
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Vascular Graft ◽  
Time Course ◽  
Platelet Reactivity ◽  
Polymer Surfaces ◽  
In Vitro Perfusion ◽  
Citrate Anticoagulation ◽  
Platelet Release

SummaryHeparin is known to affect platelet function in vitro, but little is known about the effect of heparin on the interaction of platelets with polymer surfaces in general, and vascular graft materials in particular. For this reason, the effect of heparin vs. citrate anticoagulation on the interaction of platelets with the vascular graft materials expanded polytetrafluoroethylene (ePTFE), Dacron Bionit (DB) and preclotted Dacron Bionit (DB/PC) was studied in a recirculating, in vitro perfusion system. Platelet activation, as shown by a decrease in platelet count, an increase in platelet release and a decrease in platelet aggregation, was observed for all vascular graft materials tested using heparin and was greater for Dacron and preclotted Dacron than for ePTFE. Significant differences between heparin and citrate anticoagulation were seen for platelet release, platelet aggregation and the relative ranking of material platelet-reactivity. However, the trends and time course of platelet activation were similar with both heparin and citrate for the materials tested.

Studies on the mechanism of acute inhibition of thyroglobulin hydrolysis by iodine

1985 ◽  
Vol 108 (4) ◽  
pp. 511-517 ◽  
Author(s):  
Nandalal Bagchi ◽  
Birdie Shivers ◽  
Thomas R. Brown
Keyword(s):  
Time Course ◽  
Period 2 ◽  
Iodotyrosine Deiodinase ◽  
Rate Of Hydrolysis ◽  
Underlying Mechanisms ◽  
Excess Iodide ◽  
Sites Of Action ◽  
Hydrolysis Of

Abstract. Iodine in excess is known to acutely inhibit thyroidal secretion. In the present study we have characterized the time course of the iodine effect in vitro and investigated the underlying mechanisms. Labelled thyroid glands were cultured in vitro in medium containing mononitrotyrosine, an inhibitor of iodotyrosine deiodinase. The rate of hydrolysis of labelled thyroglobulin was measured as the proportion of labelled iodotyrosines and iodothyronines recovered at the end of culture and was used as an index of thyroidal secretion. Thyrotrophin (TSH) administered in vivo acutely stimulated the rate of thyroglobulin hydrolysis. Addition of Nal to the culture medium acutely inhibited both basal and TSH-stimulated thyroglobulin hydrolysis. The effect of iodide was demonstrable after 2 h, maximal after 6 h and was not reversible upon removal of iodide. Iodide abolished the dibutyryl cAMP induced stimulation of thyroglobulin hydrolysis. Iodide required organic binding of iodine for its effect but new protein or RNA synthesis was not necessary. The inhibitory effects of iodide and lysosomotrophic agents such as NH4C1 and chloroquin on thyroglobulin hydrolysis were additive suggesting different sites of action. Iodide added in vitro altered the distribution of label in prelabelled thyroglobulin in a way that suggested increased coupling in the thyroglobulin molecule. These data indicate that 1) the iodide effect occurs progressively over a 6 h period, 2) continued presence of iodide is not necessary once the inhibition is established, 3) iodide exerts its action primarily at a post cAMP, prelysosomal site and 4) the effect requires organic binding of iodine, but not new RNA or protein synthesis. Our data are consistent with the hypothesis that excess iodide acutely inhibits thyroglobulin hydrolysis by increasing the resistance of thyroglobulin to proteolytic degradation through increased iodination and coupling.

scholarly journals DDRE-09. DEVELOPING IDO-PROTACS TO IMPROVE IMMUNOTHERAPEUTIC EFFICACY IN PATIENTS WITH GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii63-ii63
Author(s):  
Lakshmi Bollu ◽  
Derek Wainwright ◽  
Lijie Zhai ◽  
Erik Ladomersky ◽  
Kristen Lauing ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Time Course ◽  
Immune Cell ◽  
Enzyme Inhibitors ◽  
Tumor Development ◽  
Ubiquitin Proteasome System ◽  
Degradation Pathway ◽  
Specific Protein ◽  
Cell Functions

Abstract INTRODUCTION Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is a rate-limiting enzyme that metabolizes the essential amino acid tryptophan into kynurenine. Recent work by our group has revealed that IDO promotes tumor development and suppresses immune cell functions independent of its enzyme activity. Moreover, pharmacologic IDO enzyme inhibitors that currently serve as the only class of drugs available for targeting immunosuppressive IDO activity, fail to improve the survival of patients with GBM. Here, we developed IDO-Proteolysis Targeting Chimeras (IDO-PROTACs). PROTACs bind to a specific protein and recruit an E3 ubiquitin ligase that enhance proteasome-mediated degradation of the target protein. METHODS A library of ≥100 IDO-PROTACs were developed by joining BMS986205 (IDO binder) with a linker group to various E3-ligase ligands. Western blot analysis of PROTAC-induced IDO degradation was tested in vitro among multiple human and mouse GBM cell lines including U87, GBM6, GBM43 and GL261 along a time course ranging between 1–96 hours of treatment and at varying concentrations. The mechanism of IDO protein degradation was investigated using pharmacologic ligands that inhibit or compete with the proteasome-mediated protein degradation pathway. RESULTS Primary screening identified several IDO-PROTACs with IDO protein degradation potential. Secondary screening showed that our lead compound has a DC50 value of ~0.5µM with an ability to degrade IDO in all GBM cells analyzed, and an initial activity within 12 hours of treatment that extended for up to 96 hours. Mutating the CRBN-binding ligand, pretreatment with the ubiquitin proteasome system inhibitors MG132 or MLN4924 or using unmodified parental compound all inhibited IDO protein degradation. CONCLUSIONS This study developed an initial IDO-PROTAC technology that upon further optimization, can neutralize both IDO enzyme and non-enzyme immunosuppressive effects. When combined with other forms of immunotherapy, IDO-PROTACs have the potential to substantially enhance immunotherapeutic efficacy in patients with GBM.

scholarly journals Effects of tacrolimus on autophagy protein LC3 in puromycin-damaged mouse podocytes

2020 ◽  
Vol 48 (12) ◽  
pp. 030006052097142
Author(s):  
Xiao-qing Yang ◽  
Sheng-you Yu ◽  
Li Yu ◽  
Lin Ge ◽  
Yao Zhang ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Light Chain ◽  
In Vitro Model ◽  
Group Versus ◽  
Podocyte Injury ◽  
Apoptosis Rate ◽  
Protein Levels ◽  
Vitro Model ◽  
Intercellular Connections

Objective To investigate the mechanism through which tacrolimus, often used to treat refractory nephropathy, protects against puromycin-induced podocyte injury. Methods An in vitro model of puromycin-induced podocyte injury was established by dividing podocytes into three groups: controls, puromycin only (PAN group), and puromycin plus tacrolimus (FK506 group). Podocyte morphology, number, apoptosis rate and microtubule associated protein 1 light chain 3 alpha ( LC3) expression were compared. Results Puromycin caused podocyte cell body shrinkage and loose intercellular connections, but podocyte morphology in the FK506 group was similar to controls. The apoptosis rate was lower in the FK506 group versus PAN group. The low level of LC3 mRNA observed in untreated podocytes was decreased by puromycin treatment; however, levels of LC3 mRNA were higher in the FK506 group versus PAN group. Although LC3-I and LC3-II protein levels were decreased by puromycin, levels in the FK506 group were higher than the PAN group. Fewer podocyte autophagosomes were observed in the control and FK506 groups versus the PAN group. Cytoplasmic LC3-related fluorescence intensity was stronger in control and FK506 podocytes versus the PAN group. Conclusions Tacrolimus inhibited puromycin-induced mouse podocyte damage by regulating LC3 expression and enhancing autophagy.

scholarly journals Time Course of Early Events in the Action of Glucocorticoids on Rat Thymus Cells in Vitro

1973 ◽  
Vol 248 (8) ◽  
pp. 2922-2927
Author(s):  
Cornelius Hallahan ◽  
Donald A. Young ◽  
Allan Munck
Keyword(s):  
Time Course ◽  
Thymus Cells

scholarly journals Effects of Doxorubicin Delivery by Nitrogen-Doped Graphene Quantum Dots on Cancer Cell Growth: Experimental Study and Mathematical Modeling

Nanomaterials ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 140
Author(s):  
Madison Frieler ◽  
Christine Pho ◽  
Bong Han Lee ◽  
Hana Dobrovolny ◽  
Giridhar R. Akkaraju ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Cell Growth ◽  
Cancer Cell ◽  
Graphene Quantum Dots ◽  
Maximum Effect ◽  
Cancer Cell Growth ◽  
Nitrogen Doped ◽  
Nitrogen Doped Graphene ◽  
Doped Graphene

With 18 million new cases diagnosed each year worldwide, cancer strongly impacts both science and society. Current models of cancer cell growth and therapeutic efficacy in vitro are time-dependent and often do not consider the Emax value (the maximum reduction in the growth rate), leading to inconsistencies in the obtained IC50 (concentration of the drug at half maximum effect). In this work, we introduce a new dual experimental/modeling approach to model HeLa and MCF-7 cancer cell growth and assess the efficacy of doxorubicin chemotherapeutics, whether alone or delivered by novel nitrogen-doped graphene quantum dots (N-GQDs). These biocompatible/biodegradable nanoparticles were used for the first time in this work for the delivery and fluorescence tracking of doxorubicin, ultimately decreasing its IC50 by over 1.5 and allowing for the use of up to 10 times lower doses of the drug to achieve the same therapeutic effect. Based on the experimental in vitro studies with nanomaterial-delivered chemotherapy, we also developed a method of cancer cell growth modeling that (1) includes an Emax value, which is often not characterized, and (2), most importantly, is measurement time-independent. This will allow for the more consistent assessment of the efficiency of anti-cancer drugs and nanomaterial-delivered formulations, as well as efficacy improvements of nanomaterial delivery.

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