scholarly journals Development of a Kinetic Assay for Late Endosome Movement

2014 ◽  
Vol 19 (7) ◽  
pp. 1070-1078 ◽  
Author(s):  
Milan Esner ◽  
Felix Meyenhofer ◽  
Michael Kuhn ◽  
Melissa Thomas ◽  
Yannis Kalaidzidis ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Membrane Integrity ◽  
Living Cells ◽  
Mechanisms Of Action ◽  
Added Value ◽  
Kinetic Assay ◽  
Late Endosomes ◽  
Automated Imaging ◽  
Phenotypic Clustering ◽  
Large Compound

Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.

scholarly journals Exosomes induce endolysosomal permeabilization as a gateway by which exosomal tau seeds escape into the cytosol

2021 ◽  
Author(s):  
Juan Carlos Polanco ◽  
Gabriel Rhys Hand ◽  
Adam Briner ◽  
Chuanzhou Li ◽  
Jürgen Götz
Keyword(s):  
Critical Role ◽  
Membrane Integrity ◽  
Lysosomal Degradation ◽  
Escape Route ◽  
Cellular Invasion ◽  
Endosomal Membrane ◽  
Late Endosomes ◽  
Endosomal Membranes ◽  
Endosomal Pathway ◽  
Tau Aggregation

AbstractThe microtubule-associated protein tau has a critical role in Alzheimer’s disease and other tauopathies. A proposed pathomechanism in the progression of tauopathies is the trans-synaptic spreading of tau seeds, with a role for exosomes which are secretory nanovesicles generated by late endosomes. Our previous work demonstrated that brain-derived exosomes isolated from tau transgenic rTg4510 mice encapsulate tau seeds with the ability to induce tau aggregation in recipient cells. We had also shown that exosomes can hijack the endosomal pathway to spread through interconnected neurons. Here, we reveal how tau seeds contained within internalized exosomes exploit mechanisms of lysosomal degradation to escape the endosome and induce tau aggregation in the cytosol of HEK293T-derived ‘tau biosensor cells’. We found that the majority of the exosome-containing endosomes fused with lysosomes to form endolysosomes. Exosomes induced their permeabilization, irrespective of the presence of tau seeds, or whether the exosomal preparations originated from mouse brains or HEK293T cells. We also found that permeabilization is a conserved mechanism, operating in both non-neuronal tau biosensor cells and primary neurons. However, permeabilization of endolysosomes only occurred in a small fraction of cells, which supports the notion that permeabilization occurs by a thresholded mechanism. Interestingly, tau aggregation was only induced in cells that exhibited permeabilization, presenting this as an escape route of exosomal tau seeds into the cytosol. Overexpression of RAB7, which is required for the formation of endolysosomes, strongly increased tau aggregation. Conversely, inhibition of lysosomal function with alkalinizing agents, or by knocking-down RAB7, decreased tau aggregation. Together, we conclude that the enzymatic activities of lysosomes permeabilize exosomal and endosomal membranes, thereby facilitating access of exosomal tau seeds to cytosolic tau to induce its aggregation. Our data underscore the importance of endosomal membrane integrity in mechanisms of cellular invasion by misfolded proteins that are resistant to lysosomal degradation.

STUDYING THE LOCAL MECHANICAL PROPERTIES OF LIVING CELLS VIA SCANNING ION-CONDUCTING MICROSCOPY

2020 ◽  
pp. 154-156
Author(s):  
A. Vaneev ◽  
N. Savin ◽  
A. Yakovlev ◽  
A. Alova ◽  
S. Lavrushkina ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Mechanical Properties ◽  
Actin Cytoskeleton ◽  
Tumor Cells ◽  
Cell Nucleus ◽  
Living Cells ◽  
Mechanisms Of Action ◽  
Human Prostate Cancer ◽  
Before And After ◽  
Ion Conducting

The mechanical properties of PC-3 tumor cells of human prostate cancer before and after exposure to substances acting on the actin cytoskeleton, microtubules, and cell nucleus were measured by scanning ion-conducting microscopy. Changes in local mechanical properties corresponding to the mechanisms of action of these substances were found

69 REACTIVE OXYGEN SPECIES EVALUATION OF DONKEY FROZEN SEMEN ADDED TO HOMOLOGOUS SEMINAL PLASMA ON POST-THAW

2015 ◽  
Vol 27 (1) ◽  
pp. 127
Author(s):  
P. V. L. Oliveira ◽  
J. V. Oliveira ◽  
C. Ramires Neto ◽  
Y. F. R. Sancler-Silva ◽  
C. P. Freitas-Dell'aqua ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Kinetic Parameters ◽  
Seminal Plasma ◽  
Membrane Integrity ◽  
Reactive Oxygen ◽  
Computer Assisted ◽  
Oxygen Species ◽  
Inflammation Response ◽  
Frozen Semen ◽  
Uterine Inflammation

For many years the pregnancy rate of donkey frozen semen presented lower results in donkey jennies; however, a recent study showed an increase in pregnancy rates using frozen semen added to seminal plasma on post-thaw. A hypothesis for this result is the higher uterine inflammation response after breeding when using seminal plasma. The same studies demonstrated higher uterine inflammation in the presence of higher reactive oxygen species concentration. The aim of the present study was to evaluate the content of reactive oxygen species in donkey frozen semen added to homologous seminal plasma on post-thaw. Five ejaculates from each 3 donkeys were used. Semen was diluted (1 : 1) with a skim milk-based extender (Botu-SemenTM, Botupharma, Brazil). The semen was frozen with Botu-CryoTM extender (Botupharma, Brazil) in an isothermal box in straws containing 100 × 106 of total sperm. The samples were thawed at 46°C for 20 s. After this, the straws of each donkey were divided in 2 group: control group (CG), in which the semen was incubated at 37°C for 5 min, and plasma seminal group (PG), in which the semen was incubated at 37°C for 5 min with 70% of homologous seminal plasma. Sperm kinetic parameters were evaluated by computer-assisted semen analysis, and the plasma membrane integrity (propidium iodide and fluorescein isothiocyanate -PSA) and reactive oxygen species (5–6-carboxi-2,7-diclorodihidrofluoresceindiacetate) were evaluated by flow cytometer. Comparison of sperm parameters was performed by t-test. Total motility (%, CG = 75.4 ± 8.2a v. PG = 57.5 ± 16.4b), progressive motility (%, CG = 42.0 ± 8.7a v. PG = 33.3 ± 13.2b), progressive angular velocity (μm/s, CG = 95.8 ± 10.8a v. PG = 88.9 ± 10.9b), and percentage of rapid sperm (%, CG = 58.4 ± 12.5a v. PG = 41.0 ± 17.3b) were higher in CG compare with PG. No difference (P < 0.05) was observed in membrane integrity (%, CG = 20.7 ± 7.4 v. PG = 20.6 ± 7.8); however, reactive oxygen species (%, CG = 12.3 ± 10.6a v. PG = 81.8 ± 32.5b) were higher in PG. The results of this study showed that the addition of homologous seminal plasma on post-thaw decreases the sperm kinetic parameters and viability of donkey frozen semen but increases reactive oxygen species, and this may cause higher uterine inflammation response in donkey jennies and increase their fertility.

scholarly journals Effect of N-Methylacetamide Concentration and Thawing Rate on Chicken Sperm Quality after Cryopreservation

Animals ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 824
Author(s):  
Fabio Mosca ◽  
Luisa Zaniboni ◽  
Ahmad Abdel Sayed ◽  
Nicolaia Iaffaldano ◽  
Dominga Soglia ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Kinetic Parameters ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Motile Sperm ◽  
Cell Membrane Integrity ◽  
Before And After ◽  
Freezing Procedure ◽  
Thawing Rate

In seeking alternative cryoprotectants to glycerol for a reference chicken semen freezing procedure, the aim of the present study was to compare the effect of two concentrations of N-Methylacetamide (MA) and two thawing rates on the quality of frozen-thawed semen. Semen samples were diluted in Lake pre-freezing extender, including 0.1 M trehalose in presence of 6% or 9% MA, loaded into straws, frozen in nitrogen vapors, and stored in liquid nitrogen. The following thawing treatments were used: 5 °C for 100 s and 38 °C for 30 s. Sperm quality (cell membrane integrity, motility and kinetic parameters) was assessed before and after cryopreservation. The decrease of MA concentration from 9 to 6% improved sperm quality after freezing/thawing and this effect was dependent on thawing temperature. Decreasing the MA concentration from 9 to 6% improved the proportion of undamaged membrane, motile, and progressive motile sperm recovered after thawing at 5 °C for 100 s; in contrast, no effect of the MA concentration was observed thawing at 38 °C for 30 s. Therefore, the treatment with 6% MA and thawing at 5 °C for 100 s has given the best cryoprotective action. These results contribute to improve the efficacy of the current chicken semen cryopreservation procedures.

scholarly journals Protective Effects of Tropical Fruit Processing Coproducts on Probiotic Lactobacillus Strains during Freeze-Drying and Storage

2020 ◽  
Vol 8 (1) ◽  
pp. 96 ◽  
Author(s):  
Caroliny Mesquita Araújo ◽  
Karoliny Brito Sampaio ◽  
Francisca Nayara Dantas Duarte Menezes ◽  
Erika Tayse da Cruz Almeida ◽  
Marcos dos Santos Lima ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Freeze Drying ◽  
Room Temperature ◽  
Membrane Integrity ◽  
Added Value ◽  
Protective Effects ◽  
Tropical Fruit ◽  
Freeze Dried ◽  
Fruit Processing ◽  
And Storage

This study evaluated the protective effects of coproducts from agroindustrial processing of the tropical fruits acerola (Malpighia glabra L., ACE), cashew (Anacardium occidentale L., CAS), and guava (Psidium guayaba L., GUA) on the probiotics Lactobacillus paracasei L-10, Lactobacillus casei L-26, and Lactobacillus acidophilus LA-05 during freeze-drying and storage. The occurrence of damage to membrane integrity, membrane potential, and efflux activity of Lactobacillus cells after freeze-drying was evaluated by flow cytometry, and viable counts were measured immediately after freeze-drying and during 90 days of storage under refrigerated or room temperature conditions. Probiotic strains freeze-dried without substrate had the overall highest count reductions (0.5 ± 0.1 to 2.9 ± 0.3 log cycles) after freeze-drying. Probiotics freeze-dried with fruit processing coproducts had small cell subpopulations with damaged efflux activity and membrane potential. Average counts of probiotics freeze-dried with ACE, CAS, or GUA after 90 days of storage under refrigerated or room temperature were in the range of 4.2 ± 0.1 to 5.3 ± 0.2 and 2.6 ± 0.3 to 4.9 ± 0.2 log CFU/g, respectively, which were higher than those observed for strains freeze-dried without substrate. The greatest protective effects on freeze-dried probiotics were overall presented by ACE. These results revealed that ACE, CAS, and GUA can exert protective effects and increase the stability of probiotic lactobacilli during freeze-drying and storage, in addition to supporting a possible added-value destination for these agroindustrial coproducts as vehicles for probiotics and for the development of novel functional foods.

scholarly journals Conditioned Medium from Canine Amniotic Membrane-Derived Mesenchymal Stem Cells Improved Dog Sperm Post-Thaw Quality-Related Parameters

Animals ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1899
Author(s):  
Feriel Yasmine Mahiddine ◽  
Jin Wook Kim ◽  
Ahmad Yar Qamar ◽  
Jeong Chan Ra ◽  
Soo Hyun Kim ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Conditioned Medium ◽  
Amniotic Membrane ◽  
Membrane Integrity ◽  
Mitochondrial Activity ◽  
Flow Cytometry Analysis ◽  
Sperm Cryopreservation ◽  
Control Groups ◽  
Freezing Medium ◽  
And Control

This study investigated the effects of conditioned medium (CM) from canine amniotic membrane-derived MSCs (cAMSCs) on dog sperm cryopreservation. For this purpose, flow cytometry analysis was performed to characterize cAMSCs. The CM prepared from cAMSCs was subjected to proteomic analysis for the identification of proteins present in the medium. Sperm samples were treated with freezing medium supplemented with 0%, 5%, 10%, and 15% of the CM, and kinetic parameters were evaluated after 4–6 h of chilling at 4 °C to select the best concentration before proceeding to cryopreservation. Quality-related parameters of frozen–thawed sperm were investigated, including motility; kinetic parameters; viability; integrity of the plasma membrane, chromatin, and acrosome; and mitochondrial activity. The results showed that 10% of the CM significantly enhanced motility, viability, mitochondrial activity, and membrane integrity (p < 0.05); however, the analysis of chromatin and acrosome integrity showed no significant differences between the treatment and control groups. Therefore, we concluded that the addition of 10% CM derived from cAMSC in the freezing medium protected dog sperm during the cryopreservation process.

scholarly journals Mechanisms of action of Ru(ii) polypyridyl complexes in living cells upon light irradiation

2018 ◽  
Vol 54 (93) ◽  
pp. 13040-13059 ◽  
Author(s):  
Marta Jakubaszek ◽  
Bruno Goud ◽  
Stefano Ferrari ◽  
Gilles Gasser
Keyword(s):  
Living Cells ◽  
Mechanisms Of Action ◽  
Light Irradiation ◽  
Light Activation ◽  
Polypyridyl Complexes ◽  
Feature Article ◽  
Biological Studies

This feature article provides an overview of the most in-depth biological studies on Ru(ii) polypyridyl complexes upon light activation.

Biochemical properties and kinetic parameters of dihydroxyphenyialanine – 5-hydroxytryptophan decarboxylase in brain, liver, and adrenals of cat

1979 ◽  
Vol 57 (7) ◽  
pp. 1014-1018 ◽  
Author(s):  
Sylvie Bouchard ◽  
Andrée G. Roberge
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Enzyme Activity ◽  
Kinetic Parameters ◽  
Biochemical Properties ◽  
Mechanisms Of Action ◽  
Decarboxylase Activity ◽  
Peripheral Organs ◽  
Exogenous Addition ◽  
The Central Nervous System

Biochemical properties and kinetic parameters of nonpurified dihydroxyphenylaianine –5-hydroxytryptophan decarboxylase extracted from brain and two peripheral organs, liver and adrenals, were studied in the cat. This study shows that decarboxylase activity in brain is lower than in peripheral organs and that 5-hydroxytryptophan can be decarboxylated without exogenous addition of pyridoxal-5′-phosphate (PLP). However, the addition of PLP substantially increases the enzyme activity. Excess of coenzyme (>60 μM) induces inhibition in adrenals and liver but not in the central nervous system (CNS). The observed inhibition might be related to the presence of a tetrahydroisoquinoline derivative formed in the medium. Differentiation between mechanisms of action of decarboxylase in the CNS and peripheral organs is suggested.

scholarly journals Disinfectants Used in Stomatology and SARS-CoV-2 Infection

2021 ◽  
Author(s):  
Magdalena Stawarz-Janeczek ◽  
Agata Kryczyk-Poprawa ◽  
Bożena Muszyńska ◽  
Włodzimierz Opoka ◽  
Jolanta Pytko-Polończyk
Keyword(s):  
Maxillofacial Surgery ◽  
Membrane Integrity ◽  
Mechanisms Of Action ◽  
Sodium Chlorite ◽  
Dental Practice ◽  
Fungal Cell ◽  
Cell Membrane Integrity ◽  
Cell Components ◽  
Activity Loss ◽  
Number Of Publications

AbstractEffective disinfection is a basic procedure in medical facilities, including those conducting dental surgeries, where treatments for tissue discontinuity are also performed, as it is an important element of infection prevention. Disinfectants used in dentistry and dental and maxillofacial surgery include both inorganic (hydrogen peroxide, sodium chlorite-hypochlorite) and organic compounds (ethanol, isopropanol, peracetic acid, chlorhexidine, eugenol). Various mechanisms of action of disinfectants have been reported, which include destruction of the structure of bacterial and fungal cell membranes; damage of nucleic acids; denaturation of proteins, which in turn causes inhibition of enzyme activity; loss of cell membrane integrity; and decomposition of cell components. This article discusses the most important examples of substances used as disinfectants in dentistry and presents the mechanisms of their action with particular focus on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The search was conducted in ScienceDirect, PubMed, and Scopus databases. The interest of scientists in the use of disinfectants in dental practice is constantly growing, which results in the increasing number of publications on disinfection, sterilization, and asepsis. Many disinfectants often possess several of the abovementioned mechanisms of action. In addition, disinfectant preparations used in dental practice either contain one compound or are frequently a mixture of active compounds, which increases their range and effectiveness of antimicrobial action. Currently available information on disinfectants that can be used to prevent SARS-CoV-2 infection in dental practices was summarized.

scholarly journals Sorting and cryopreservation of goat sperm with or without phenolic compounds

2020 ◽  
Vol 72 (2) ◽  
pp. 295-304
Author(s):  
E.C.B. Silva ◽  
J.I.T. Vieira ◽  
I.H.A.V. Nery ◽  
R.A.J. Araújo Silva ◽  
V.F.M.H. Lima ◽  
...  
Keyword(s):  
Membrane Integrity ◽  
Living Cells ◽  
Phenolic Antioxidants ◽  
Density Gradients ◽  
Continuous Density ◽  
Progressive Motility ◽  
Head Displacement ◽  
Straight Line ◽  
Sperm Sorting ◽  
Lateral Head

ABSTRACT The objectives of this study were to evaluate goat sperm sorting in continuous Percoll® density gradients and gamete freezability, in the presence or absence of phenolic antioxidants. For this, semen pools were sorted, frozen, and evaluated. The non-selected group (NSg) presented lower progressive motility (PM), linearity (LIN), straightness (STR), and wobble (WOB) than the selected groups, and straight line velocity (VSL) compared to those with catechin or resveratrol. The amplitude of lateral head displacement (ALH) was higher in NSg, and quercetin reduced the mitochondrial membrane potential (MMP). After thawing, the NSg presented lower PM than the selected groups, VSL and VAP (average path velocity) than the selected group with or without catechin, LIN and WOB than the selected with or without catechin or resveratrol, and STR than the selected with catechin. Moreover, NSg presented higher ALH and BCF than the samples selected with or without catechin. Plasma membrane integrity and intact and living cells were higher in the selected groups, and MMP was lower in the NSg and the selected group with quercetin. Thus, centrifugation in Percoll® continuous density gradients is a viable methodology to select goat sperm compatible with the freezing, especially in the presence of catechin or resveratrol.

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