scholarly journals Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Annamaria Nigro ◽  
Annamaria Finardi ◽  
Marzia M. Ferraro ◽  
Daniela E. Manno ◽  
Angelo Quattrini ◽  
...  
Keyword(s):  
Common Mechanism ◽  
Differential Centrifugation ◽  
Selective Loss ◽  
Pathological Conditions ◽  
Centrifugation Step ◽  
Centrifugation Method ◽  
Downstream Analysis ◽  
Cell Conditioned Medium ◽  
Rotor Type ◽  
Centrifugation Protocol

AbstractMicrovesicles (MVs) are large extracellular vesicles differing in size, cargo and composition that share a common mechanism of release from the cells through the direct outward budding of the plasma membrane. They are involved in a variety of physiological and pathological conditions and represent promising biomarkers for diseases. MV heterogeneity together with the lack of specific markers had strongly hampered the development of effective methods for MV isolation and differential centrifugation remains the most used method to purify MVs. In this study, we analysed the capacity of the differential centrifugation method to isolate MVs from cell-conditioned medium using flow cytometry and TEM/AFM microscopy. We found that the loss of MVs (general population and/or specific subpopulations) represents a major and underestimate drawback of the differential centrifugation protocol. We demonstrate that the choice of the appropriate rotor type (fixed-angle vs swinging-bucket) and the implementation of an additional washing procedure to the first low-speed centrifugation step of the protocol allow to overcome this problem increasing the total amount of isolated vesicles and avoiding the selective loss of MV subpopulations. These parameters/procedures should be routinely employed into optimized differential centrifugation protocols to ensure isolation of the high-quantity/quality MVs for the downstream analysis/applications.

scholarly journals Circadian Deregulation as Possible New Player in Pollution-Induced Tissue Damage

Atmosphere ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 116
Author(s):  
Mascia Benedusi ◽  
Elena Frigato ◽  
Cristiano Bertolucci ◽  
Giuseppe Valacchi
Keyword(s):  
Cardiovascular Diseases ◽  
Air Pollutants ◽  
Environmental Stressors ◽  
Common Mechanism ◽  
Particulate Matters ◽  
Circadian Misalignment ◽  
Pathological Conditions ◽  
Peripheral Clocks ◽  
Living Organisms ◽  
Almost All

Circadian rhythms are 24-h oscillations driven by a hypothalamic master oscillator that entrains peripheral clocks in almost all cells, tissues and organs. Circadian misalignment, triggered by industrialization and modern lifestyles, has been linked to several pathological conditions, with possible impairment of the quality or even the very existence of life. Living organisms are continuously exposed to air pollutants, and among them, ozone or particulate matters (PMs) are considered to be among the most toxic to human health. In particular, exposure to environmental stressors may result not only in pulmonary and cardiovascular diseases, but, as it has been demonstrated in the last two decades, the skin can also be affected by pollution. In this context, we hypothesize that chronodistruption can exacerbate cell vulnerability to exogenous damaging agents, and we suggest a possible common mechanism of action in deregulation of the homeostasis of the pulmonary, cardiovascular and cutaneous tissues and in its involvement in the development of pathological conditions.

scholarly journals Non-contact acoustic capture of microparticles from small plasma volumes

Lab on a Chip ◽  
2015 ◽  
Vol 15 (12) ◽  
pp. 2588-2596 ◽  
Author(s):  
Mikael Evander ◽  
Olof Gidlöf ◽  
Björn Olde ◽  
David Erlinge ◽  
Thomas Laurell
Keyword(s):  
Contact Method ◽  
Differential Centrifugation ◽  
Limited Volume ◽  
Centrifugation Method

An acoustic non-contact method for extracting platelet-derived microparticles from small plasma volumes is presented. The method has higher recovery and can handle smaller sample volumes than conventional differential centrifugation method. It is optimal for the analysis of precious biobank samples with limited volume.

scholarly journals Discrete-Impulse Energy Supply in Milk and Dairy Product Processing

Materials ◽  
2021 ◽  
Vol 14 (15) ◽  
pp. 4181
Author(s):  
Paweł Droździel ◽  
Tetiana Vitenko ◽  
Viktor Voroshchuk ◽  
Sergiy Narizhnyy ◽  
Olha Snizhko
Keyword(s):  
Milk Fat ◽  
Energy Supply ◽  
Dairy Product ◽  
Dynamic Effect ◽  
Fat Emulsion ◽  
Cream Cheese ◽  
Effective Change ◽  
Centrifugation Method ◽  
Rotor Type ◽  
Type Apparatus

The efficient use of supplied energy is the basis of the discrete-impulse energy supply (DIES) concept. In order to explore the possibility of using DIES to intensify the hydromechanical processes, the emulsification of milk fat (homogenization of milk, preparation of spreads) and, in particular, the processing of cream cheese masses, were studied. Whole non-homogenized milk, fat emulsions, and cream cheese mass were the object of investigation. To evaluate the efficiency of milk homogenization, the homogenization coefficient change was studied, which was determined by using the centrifugation method, as it is the most affordable and accurate one. To provide the proper dispersion of the milk emulsion, six treatment cycles must be carried out under the developed cavitation mode in a static-type apparatus, here resulting in a light grain-like consistency, and exhibiting the smell of pasteurized milk. The emulsions were evaluated according to the degree of destabilization, resistance and dispersion of the fat phase. On the basis of the obtained data with respect to the regularities of fat dispersion forming in the rotor-type apparatus, the proper parameters required to obtain technologically stable fat emulsion spreads, possessing a dispersion and stability similar to those of plain milk creams, were determined. It was determined that under the DIES, an active dynamic effect on the milk globules takes place. The rheological characteristics of cheese masses were evaluated on the basis of the effective change in viscosity. The effect of the mechanical treatment on the structure of the cheese masses was determined. 

Radioimmunoassay of human thyroglobulin with use of "thyroglobulin-free" plasma prepared by ultracentrifugation as diluent.

1980 ◽  
Vol 26 (11) ◽  
pp. 1566-1568 ◽  
Author(s):  
K Ikekubo ◽  
J Jutton ◽  
A B Schneider
Keyword(s):  
Total Thyroidectomy ◽  
Human Plasma ◽  
Normal Subjects ◽  
New Method ◽  
Differential Centrifugation ◽  
Thyroid Malignancy ◽  
Centrifugation Method ◽  
Preparation And Evaluation ◽  
Free Plasma

Abstract We describe the preparation and evaluation of a new diluent for use in preparing standard curves for the radioimmunoassay of human thyroglobulin (TG). This diluent, "TG-free human plasma, allows measurement of TG in lower concentrations than with previous assay systems because the components of the standards are more similar to those of the samples. TG-free plasma was prepared by a modification of the differential centrifugation method of Edelhoch (J. Biol. Chem. 235:1326, 1960). Standard curves prepared by use of TG-free plasma and plasma obtained from thyroidectomized patients were identical. In our assay, plasma from patients who had undergone total thyroidectomy produced no displacement when 25 to 100 microL of plasma was assayed. Twenty-five normal subjects all had detectable TG concentrations, ranging from 4.0 to 25.7 microgram/L. Only 27 of 2000 determinations performed by this new method had undetectable values and 26 of these were from patients with total or near-total thyroidectomies. TG concentration determined by this assay may be more sensitive for finding recurrent thyroid malignancy.

Beneficial Role of Tetrahydrobiopterin on Various Cardiovascular Diseases and Regulation of its Levels under Pathological Conditions

Pteridines ◽  
2006 ◽  
Vol 17 (1) ◽  
pp. 5-10
Author(s):  
Shunichi Shimizu ◽  
Masakazu Ishii ◽  
Teruaki Wajima ◽  
Tamio Hagiwara ◽  
Takaharu Negoro
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Cardiovascular Diseases ◽  
Reperfusion Injury ◽  
Vascular Endothelial Cells ◽  
Ischemia Reperfusion ◽  
Vascular Diseases ◽  
Common Mechanism ◽  
Pathological Conditions

Abstract 5,6,7,8-Tetrahydrobiopterin (BH4) is an csscntial cofactor for production of nitric oxide (NO) by N O synthase (NOS). Nitric oxide (NO) is an important signaling mo!ecu!e for the regulation o f vita! functions such as vascular tone, neurotransmission and immune surveiüance. Accumu!ated evidences show that although NOS mainly re!eases N O under nonnal conditions, NOS also produces Superoxide anion and hydrogen peroxide when BH4 is decreased, suggesting the possibility that NOS is a source of reactive oxygen species (ROS) under pathological conditions. In fact, supplementation of BH4 restores N O production and availability in various diseases including hyperlipidemia, diabetes, hypertension and ischemia-reperfusion. Moreover, recent studies show that administration o f BH4 protects tissues against ischemia-reperfusion injuiy in heart, stomach and liver. Oxidative stress is a common mechanism underlying the development of vascular diseases and reperfusion injury. Regulation of BH4 levels under oxidative stress is important to understand the role of BH4 on the development of vascular diseases and reperfusion injury. We recently described that oxidative stress transiently decreased BH4 levels, and then markedly increased its levels in vascular endothelial cells. Supplementation of BH4 appears to be important therapeutic strategies in vascular diseases and reperfusion injury, and induction of BH4 synthesis may endogenous defense system against oxidative stress-mediated cardiovascular diseases.

scholarly journals A Review on the Neuroendocrine Regulation of the Endothelial Cells

2020 ◽  
pp. 1-4
Author(s):  
Paolo Lissoni ◽  
◽  
Franco Rovelli ◽  
Keyword(s):  
Neuroendocrine System ◽  
Neuroendocrine Regulation ◽  
Common Mechanism ◽  
Systemic Diseases ◽  
Cytokine Network ◽  
Pathological Conditions ◽  
Inflammatory Processes ◽  
The Common ◽  
Endothelial Functions ◽  
Inflammatory Action

Today, it is known that inflammation represents the common mechanism of human systemic diseases, including cancer and autoimmunity. Obviously, the endothelial system is involved in all inflammatory processes. Then, the control of the endothelial functions could constitute a new medical strategy to treat several pathological conditions, including ischemic and thrombotic events. Moreover, in addition to the action of angiogenic and anti-angiogenic factors, the endothelial system has been proven to be physiologically under a double control, represent by the cytokine network and the neuroendocrine system. Most cytokines have appeared to exert angiogenic and inflammatory effects, which are balanced by an anti-angiogenic and an anti-inflammatory action exerted by the pineal hormone melatonin (MLT), cannabinoid agents, and the product of ACE2, the angiotensin 1-7 (Ang 1-7). Then, a neuroendocrine approach with MLT, cannabinoids and Ang 1-7 could constitute a new way in the treatment of endothelial alterations and angiogenesis.

scholarly journals Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Mikhail A. Livshits ◽  
Elena Khomyakova ◽  
Evgeniy G. Evtushenko ◽  
Vassili N. Lazarev ◽  
Nikolay A. Kulemin ◽  
...  
Keyword(s):  
Culture Supernatant ◽  
Therapeutic Agents ◽  
Ht29 Cell ◽  
Cell Culture Supernatant ◽  
Nanoparticle Tracking Analysis ◽  
Differential Centrifugation ◽  
Fixed Angle ◽  
Disease Markers ◽  
Path Lengths ◽  
Centrifugation Protocol

Abstract Exosomes, small (40–100 nm) extracellular membranous vesicles, attract enormous research interest because they are carriers of disease markers and a prospective delivery system for therapeutic agents. Differential centrifugation, the prevalent method of exosome isolation, frequently produces dissimilar and improper results because of the faulty practice of using a common centrifugation protocol with different rotors. Moreover, as recommended by suppliers, adjusting the centrifugation duration according to rotor K-factors does not work for “fixed-angle” rotors. For both types of rotors – “swinging bucket” and “fixed-angle” – we express the theoretically expected proportion of pelleted vesicles of a given size and the “cut-off” size of completely sedimented vesicles as dependent on the centrifugation force and duration and the sedimentation path-lengths. The proper centrifugation conditions can be selected using relatively simple theoretical estimates of the “cut-off” sizes of vesicles. Experimental verification on exosomes isolated from HT29 cell culture supernatant confirmed the main theoretical statements. Measured by the nanoparticle tracking analysis (NTA) technique, the concentration and size distribution of the vesicles after centrifugation agree with those theoretically expected. To simplify this “cut-off”-size-based adjustment of centrifugation protocol for any rotor, we developed a web-calculator.

scholarly journals A Review on the Neuroendocrine Regulation of the Endothelial Cells

2021 ◽  
pp. 1-4
Author(s):  
Paolo Lissoni ◽  
◽  
Franco Rovelli ◽  
Giusy Messina ◽  
◽  
...  
Keyword(s):  
Neuroendocrine System ◽  
Neuroendocrine Regulation ◽  
Common Mechanism ◽  
Systemic Diseases ◽  
Cytokine Network ◽  
Pathological Conditions ◽  
Inflammatory Processes ◽  
The Common ◽  
Endothelial Functions ◽  
Inflammatory Action

Today, it is known that inflammation represents the common mechanism of human systemic diseases, including cancer and autoimmunity. Obviously, the endothelial system is involved in all inflammatory processes. Then, the control of the endothelial functions could constitute a new medical strategy to treat several pathological conditions, including ischemic and thrombotic events. Moreover, in addition to the action of angiogenic and anti-angiogenic factors, the endothelial system has been proven to be physiologically under a double control, represent by the cytokine network and the neuroendocrine system. Most cytokines have appeared to exert angiogenic and inflammatory effects, which are balanced by an anti-angiogenic and an anti-inflammatory action exerted by the pineal hormone melatonin (MLT), cannabinoid agents, and the product of ACE2, the angiotensin 1-7 (Ang 1-7). Then, a neuroendocrine approach with MLT, cannabinoids and Ang 1-7 could constitute a new way in the treatment of endothelial alterations and angiogenesis.

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