scholarly journals A two-step model of autophagy: autophagosome formation, degradation and net turnover

2020 ◽  
Author(s):  
Ainhoa Plaza-Zabala ◽  
Virginia Sierra-Torre ◽  
Amanda Sierra
Keyword(s):  
Western Blot ◽  
Autophagic Flux ◽  
Autophagosome Formation ◽  
Autophagy Flux ◽  
Gold Standard Method ◽  
Complex Process ◽  
Step Model ◽  
Using Data ◽  
Definition Of ◽  
The Brain

AbstractAutophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. A gold-standard method to assess its induction is the analysis of the autophagic flux using as a surrogate the expression of the microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine (LC3-II) by Western blot, in the presence of lysosomal inhibitors. Therefore, the current definition of autophagy flux actually puts the focus on the degradation stage of autophagy. In contrast, the most important autophagy controlling genes that have been identified in the last few years in fact target early stages of autophagosome formation. From a biological standpoint is therefore conceivable that autophagosome formation and degradation are independently regulated and we argue that both stages need to be systematically analyzed. Here, we propose a simple two-step model to understand changes in autophagosome formation and degradation using data from conventional LC3-II Western blot, and test it using two models of autophagy modulation in cultured microglia, the brain macrophages: rapamycin and the ULK1/2 inhibitor, MRT68921. Our model provides a comprehensive understanding of the autophagy process and will help to unravel the effect of genetic, pharmacological, and environmental manipulations on both the formation and degradation of autophagosomes.

scholarly journals Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover

2021 ◽  
Vol 11 ◽  
Author(s):  
Ainhoa Plaza-Zabala ◽  
Virginia Sierra-Torre ◽  
Amanda Sierra
Keyword(s):  
Western Blot ◽  
Cell Types ◽  
Autophagic Flux ◽  
Immune Functions ◽  
Autophagosome Formation ◽  
Gold Standard Method ◽  
Complex Process ◽  
Step Model ◽  
Using Data ◽  
Definition Of

Autophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. Autophagy is increasingly recognized as a critical process in macrophages, including microglia, as it finely regulates innate immune functions such as inflammation. A gold-standard method to assess its induction is the analysis of the autophagic flux using as a surrogate the expression of the microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine (LC3-II) by Western blot, in the presence of lysosomal inhibitors. Therefore, the current definition of autophagy flux actually puts the focus on the degradation stage of autophagy. In contrast, the most important autophagy controlling genes that have been identified in the last few years in fact target early stages of autophagosome formation. From a biological standpoint is therefore conceivable that autophagosome formation and degradation are independently regulated and we argue that both stages need to be systematically analyzed. Here, we propose a simple two-step model to understand changes in autophagosome formation and degradation using data from conventional LC3-II Western blot, and test it using two models of autophagy modulation in cultured microglia: rapamycin and the ULK1/2 inhibitor, MRT68921. Our two-step model will help to unravel the effect of genetic, pharmacological, and environmental manipulations on both formation and degradation of autophagosomes, contributing to dissect out the role of autophagy in physiology and pathology in microglia as well as other cell types.

scholarly journals Beclin 1, LC3 and P62 Expression in Equine Sarcoids

Animals ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 20
Author(s):  
Manuela Martano ◽  
Gennaro Altamura ◽  
Karen Power ◽  
Pierluigi Liguori ◽  
Brunella Restucci ◽  
...  
Keyword(s):  
Western Blot ◽  
Normal Skin ◽  
Beclin 1 ◽  
Autophagic Flux ◽  
Damaged Material ◽  
Complex Process ◽  
Causative Agents ◽  
No Activation ◽  
Skin Samples ◽  
Selection Of

Background: It is well known that δ-bovine papillomaviruses (BPV-1, BPV-2 and BPV-13) are one of the major causative agents of equine sarcoids, the most common equine skin tumors. Different viruses, including papillomaviruses, evolved ingenious strategies to modulate autophagy, a complex process involved in degradation and recycling of old and damaged material. Methods: The aim of this study was to evaluate, by immunohistochemistry (IHC) and Western blot (WB) analysis, the expression of the main related autophagy proteins (Beclin 1, protein light chain 3 (LC3) and P62), in 35 BPV1/2 positive equine sarcoids and 5 BPV negative normal skin samples. Results: Sarcoid samples showed from strong-to-moderate cytoplasmic immunostaining, respectively, for Beclin 1 and P62 in >60% of neoplastic fibroblasts, while LC3 immunostaining was weak to moderate in ≤60% of neoplastic fibroblasts. Western blot analysis confirmed the specificity of the antibodies and revealed no activation of autophagic flux despite Beclin 1 overexpression in sarcoid samples. Conclusion: Results could suggest the activation of the initial phase of autophagy in equine sarcoids, and its impairment during the following steps. The impairment of autophagy could lead to a selection of a quiescent population of fibroblasts, which survive longer in a hypoxic microenvironment and produced more and/or altered collagen.

scholarly journals Apelin Promotes ECM Synthesis by Enhancing Autophagy Flux via TFEB in Human Degenerative NP Cells under Oxidative Stress

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Wei Jiang ◽  
Pei Zhao ◽  
Xuemei Zhang
Keyword(s):  
Oxidative Stress ◽  
Western Blot ◽  
Underlying Mechanism ◽  
Autophagic Flux ◽  
Rt Pcr ◽  
Autophagy Flux ◽  
Protein Expressions ◽  
Collagen Ii ◽  
The Relationship ◽  
And Oxidative Stress

Background. Apelin alleviates oxidative stress which contributes to the development of aging. IVDD is a disease closely correlated to aging and oxidative stress which is known to be harmful to NP cells’ matrix synthesis. The purpose of the present study was to investigate the role and underlying mechanism of Apelin in NP cells’ matrix degradation under oxidative stress. Methods. First, the mRNA and protein expressions of Apelin were checked by RT-PCR and Western blot in NP from normal and degenerative IVD to explore the relationship between Apelin and IVDD preliminarily. Then, H2O2 was used to mimic oxidative stress of NP cells. After treated with Apelin 13 and CQ, the GAG content was assessed by DMMB and the mRNA/protein expressions of NP matrix macromolecules (Collagen II and Aggrecan) and autophagy-related markers (LC3 and p62) were assessed by RT-PCR/Western blot. Finally, TFEB was knocked down by esiRNA-TFEB transfection and the nucleoprotein expression of TFEB and autophagy-related markers (LC3 and p62) were assessed by Western blot to discuss whether TFEB is involved in Apelin regulating autophagy flux in NP cells under oxidative stress. Results. Our data first confirmed that the mRNA and protein expressions of Apelin were decreased with IVDD. Furthermore, Apelin increased GAG content of NP cells and mRNA/protein expressions of NP matrix macromolecules (Collagen II and Aggrecan) and promoted autophagic flux (LC3II/I increased and p62 decreased) under oxidative stress. Finally, after transfected with esiRNA-TFEB, Apelin cannot promote autophagic flux any more in human degenerative NP cells. Conclusion. Our data indicated that Apelin promotes ECM synthesis by enhancing autophagy flux via TFEB in human degenerative NP cells under oxidative stress. This viewpoint may provide a new therapeutic idea for IVDD.

scholarly journals Activation of CD137 Signaling Enhances Vascular Calcification through c-Jun N-Terminal Kinase-Dependent Disruption of Autophagic Flux

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Rui Chen ◽  
Yao Xu ◽  
Wei Zhong ◽  
Bo Li ◽  
Ping Yang ◽  
...  
Keyword(s):  
Western Blot ◽  
Vascular Calcification ◽  
Underlying Mechanism ◽  
Calcium Deposition ◽  
Control Group ◽  
Autophagic Flux ◽  
Autophagy Flux ◽  
Alp Activity

Background. Vascular calcification is widespread and clinically significant, contributing to substantial morbidity and mortality. Calcifying vascular cells are partly derived from local vascular smooth muscle cells (VSMCs), which can undergo chondrogenic or osteogenic differentiation under inflammatory environment. Recently, we have found activation of CD137 signaling accelerated vascular calcification. However, the underlying mechanism remains unknown. This study aims to identify key mediators involved in CD137 signaling-induced vascular calcification in vivo and in vitro. Methods. Autophagy flux was measured through mRFP-GFP-LC3 adenovirus and transmission electron microscopy. Von Kossa assay and alkaline phosphatase (ALP) activity were used to observe calcification in vivo and in vitro, respectively. Autophagosome-containing vesicles were collected and identified by flow cytometry and Western blot. Autophagy or calcification-associated targets were measured by Western blot, quantitative real-time PCR, and immunohistochemistry. Results. Treatment with the agonist-CD137 displayed c-Jun N-terminal kinase- (JNK-) dependent increase in the expression of various markers of autophagy and the number of autophagosomes relative to the control group. Autophagy flux experiments suggested that agonist-CD137 blocked the fusion of autophagosomes with lysosomes in cultured VSMCs. Calcium deposition, ALP activity, and the expression of calcification-associated proteins also increased in agonist-CD137 group compared with anti-CD137 group, which could be recovered by autophagy stimulator rapamycin. Autophagosome-containing vesicles collected from agonist-CD137 VSMCs supernatant promoted VSMC calcification. Conclusion. The present study identified a new pathway in which CD137 promotes VSMC calcification through the activation of JNK signaling, subsequently leading to the disruption of autophagic flux, which is responsible for CD137-induced acceleration of vascular calcification.

scholarly journals Why to Study the Brain

2019 ◽  
Vol 1 (2) ◽  
pp. 1-2
Author(s):  
Ruggero Micheletto
Keyword(s):  
Computer Algorithm ◽  
Computational Process ◽  
Complex Process ◽  
Definition Of ◽  
The Mind ◽  
The Brain ◽  
The Universe

The brain is the ultimate computational machine in the universe. Despite what was thought about the brain decades and centuries ago, now it is well understood that the brain is a calculator, a machine that does computational things. In other words the brain is something that takes information in its input, elaborates it and then spit-out some result in form of information or actions. Many centuries ago, the brain was the location of the mind. The mind was considered like a mysterious entity where our thought, our consciousness and our feeling resided. The definition of mind of course is still valid today, in some sense. What has changed is the fact that now we know that the mind is the results of a computational process. A very, complex process, presumably a stochastic one, but as far as we know, something that we can define as a computer “algorithm” is running inside our brains. Data are processes in a way that is not yet completely clear, and the result is our entire behavior, our thoughts our actions and our perception of reality

Application of Scanning Electron Microscopy to Medical Research

1969 ◽  
Vol 27 ◽  
pp. 12-13
Author(s):  
J. D. Hutchison
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Medical Research ◽  
Prosthetic Heart Valve ◽  
School Of Medicine ◽  
Transmission Electron ◽  
Definition Of ◽  
Mechanism Of Failure ◽  
The Brain ◽  
Scanning Electron

When the transmission electron microscope was commercially introduced a few years ago, it was heralded as one of the most significant aids to medical research of the century. It continues to occupy that niche; however, the scanning electron microscope is gaining rapidly in relative importance as it fills the gap between conventional optical microscopy and transmission electron microscopy.IBM Boulder is conducting three major programs in cooperation with the Colorado School of Medicine. These are the study of the mechanism of failure of the prosthetic heart valve, the study of the ultrastructure of lung tissue, and the definition of the function of the cilia of the ventricular ependyma of the brain.

Comparison of Fluorine(18)-fluorodeoxyglucose and Gallium(68)-citrate PET/CT in patients with tuberculosis

2019 ◽  
Vol 58 (05) ◽  
pp. 371-378
Author(s):  
Alfred O. Ankrah ◽  
Ismaheel O. Lawal ◽  
Tebatso M.G. Boshomane ◽  
Hans C. Klein ◽  
Thomas Ebenhan ◽  
...  
Keyword(s):  
Tracer Uptake ◽  
Pet Ct ◽  
The Mean ◽  
18F Fdg ◽  
Definition Of ◽  
Gallium 68 ◽  
Attending Physician ◽  
Pet Scans ◽  
The Brain

Abstract 18F-FDG and 68Ga-citrate PET/CT have both been shown to be useful in the management of tuberculosis (TB). We compared the abnormal PET findings of 18F-FDG- and 68Ga-citrate-PET/CT in patients with TB. Methods Patients with TB on anti-TB therapy were included. Patients had a set of PET scans consisting of both 18F-FDG and 68Ga-citrate. Abnormal lesions were identified, and the two sets of scans were compared. The scan findings were correlated to the clinical data as provided by the attending physician. Results 46 PET/CT scans were performed in 18 patients, 11 (61 %) were female, and the mean age was 35.7 ± 13.5 years. Five patients also had both studies for follow-up reasons during the use of anti-TB therapy. Thirteen patients were co-infected with HIV. 18F-FDG detected more lesions than 68Ga-citrate (261 vs. 166, p < 0.0001). 68Ga-citrate showed a better definition of intracerebral lesions due to the absence of tracer uptake in the brain. The mean SUVmax was higher for 18F-FDG compared to 68Ga-citrate (5.73 vs. 3.01, p < 0.0001). We found a significant correlation between the SUVmax of lesions that were determined by both tracers (r = 0.4968, p < 0.0001). Conclusion Preliminary data shows 18F-FDG-PET detects more abnormal lesions in TB compared to 68Ga-citrate. However, 68Ga-citrate has better lesion definition in the brain and is therefore especially useful when intracranial TB is suspected.

DIRECTIONS OF ASSOCIATIVE IDENTIFICATION OF PERSONAL NAMES

2019 ◽  
Vol 18 (28) ◽  
pp. 118-128
Author(s):  
Olena Karpenko ◽  
Tetiana Stoianova
Keyword(s):  
Proper Names ◽  
Mental Lexicon ◽  
Point Of View ◽  
Sound Symbolism ◽  
Personal Names ◽  
Communicative Process ◽  
Proper Name ◽  
Definition Of ◽  
Scientific Value ◽  
The Brain

The article is devoted to the study of personal names from a cognitive point of view. The study is based on the cognitive concept that speech actually exists not in the speech, not in linguistic writings and dictionaries, but in consciousness, in the mental lexicon, in the language of the brain. The conditions for identifying personal names can encompass not only the context, encyclopedias, and reference books, but also the sound form of the word. In the communicative process, during a free associative experiment, which included a name and a recipient’s mental lexicon. The recipient was assigned a task to quickly give some association to the name. The aggregate of a certain number of reactions of different recipients forms the associative field of a proper name. The associative experiment creates the best conditions for identifying the lexeme. The definition of a monosemantic personal name primarily includes the search of what it denotes, while during the process of identifying a polysemantic personal name recipients tend have different reactions. Scientific value is posed by the effect of the choice of letters for the name, sound symbolism, etc. The following belong to the generalized forms of identification: usage of a hyperonym; synonyms and periphrases or simple descriptions; associations denoting the whole (name stimulus) by reference to its part (associatives); cognitive structures such as “stimulus — association” and “whole (stimulus) — part (associative)”; lack of adjacency; mysterious associations. The topicality of the study is determined by its perspective to identify the directions of associative identification of proper names, which is one of the branches of cognitive onomastics. The purpose of the study is to identify, review, and highlight the directions of associative identification of proper names; the object of the research is the names in their entirety and variety; its subject is the existence of names in the mental lexicon, which determines the need for singling out the directions for the associative identification of the personal names.

Neuroethics

2017 ◽  
Keyword(s):  
Brain Function ◽  
Ethical Issues ◽  
Wearable Technologies ◽  
Definition Of Death ◽  
Populations At Risk ◽  
The Future ◽  
Aging Adults ◽  
Definition Of ◽  
The Brain ◽  
New Criteria

We have new answers to how the brain works and tools which can now monitor and manipulate brain function. Rapid advances in neuroscience raise critical questions with which society must grapple. What new balances must be struck between diagnosis and prediction, and invasive and noninvasive interventions? Are new criteria needed for the clinical definition of death in cases where individuals are eligible for organ donation? How will new mobile and wearable technologies affect the future of growing children and aging adults? To what extent is society responsible for protecting populations at risk from environmental neurotoxins? As data from emerging technologies converge and are made available on public databases, what frameworks and policies will maximize benefits while ensuring privacy of health information? And how can people and communities with different values and perspectives be maximally engaged in these important questions? Neuroethics: Anticipating the Future is written by scholars from diverse disciplines—neurology and neuroscience, ethics and law, public health, sociology, and philosophy. With its forward-looking insights and considerations for the future, the book examines the most pressing current ethical issues.

Sign in / Sign up

Export Citation Format

Share Document