RT @: Depletion of abundant #plasma #proteins for #extracellular #vesicle proteome characterization: benefits and pitfalls
by Sandrine Reymond et al @UNIGEnews @unige_en #proteomics #chromatography #EVs #biomarker

#OpenAccess
https://link.springer.com/article/10.1007/s00216-023-04684-w…

@VineyTJ
“ My favourite technique is in vivo #extracellular recordings and #juxtacellular labelling” that flex 💅​
(Not a criticism - I’m just impressed)

Welcome!!

New #introduction for neuromatch.social 🙂​

My group investigates and defines cell types and neural circuits of the #thalamus, #hippocampus, and #cortex that contribute to spatial #memory processes.
These processes break down in #Alzheimer's disease. To understand the causes/triggers of #neurodegeneration, we study early-stage #Tau #pathology in the human #brain and in mouse models.

I previously defined various types of retinal ganglion cells using ex vivo #patchclamp recordings during my PhD in #Basel, before moving to #Oxford to work on #GABAergic #neurons of the #hippocampus, followed by the #medialseptum. My favourite technique is in vivo #extracellular recordings and #juxtacellular labelling as it enables identification of single #cells based on their firing patterns, #axon terminal distribution, and #neurochemical profile.

As an #experimentalist, my expertise is primarily in vivo #neurophysiology and #neuroanatomy. I am also interested in #consciousness, the origins of #memory, and regulation of #neuronal activity and #behaviour by #neuropeptides.

This is because the tissue-engineered constructs do not have the same mechanical properties as native articular cartilage, which is due to the insufficient accumulation of #extracellular matrix components. To address this, researchers have been exploring the use of adenosine triphosphate (#ATP) to directly harness the underlying mechanotransduction pathways responsible.

#Articular #cartilage, which is a type of #tissue found in #joints, allows for nearly frictionless motion and can absorb large loads. Unfortunately, when it is damaged, it cannot repair itself. #Tissueengineering is a promising approach to repair the damage, but it falls short of creating functional tissue. This is because the tissue-engineered constructs do not have the same mechanical properties as native articular cartilage, which is due to the insufficient accumulation of #extracellular matrix components. To address this, researchers have been exploring the use of adenosine triphosphate (#ATP) to directly harness the underlying mechanotransduction pathways responsible. ATP is a molecule that is released as a result of mechanical stimulation and acts as an autocrine/paracrine signaling #molecule. It acts on P2 receptors on the #plasma #membrane to promote extracellular matrix #synthesis. However, high doses of ATP can lead to an increase in matrix #metalloproteinase 13 (MMP-13) activity and extracellular inorganic pyrophosphate (ePPi) accumulation, which can lead to undesirable effects such as #mineralization of articular cartilage. Therefore, the purpose of this study is to identify the mechanism of ATP-mediated #catabolism and to determine a therapeutic dose range to maximize the #anabolic effect.

Materials & Methods

Cell Isolation: This is the process of separating cells from a tissue sample. It is usually done using #enzymes to break down the tissue and then filtering the cells out.

3-Dimensional Culture: This is a type of #cellculture where the cells are grown in a three-dimensional environment, rather than in a flat layer. This allows the cells to interact with each other in a more natural way.

Exogenous ATP Supplementation: ATP (adenosine triphosphate) is a molecule that is important for energy production in cells. Exogenous ATP supplementation is the process of adding ATP to the cell culture from an outside source. This can help the cells to grow and function better.

MMP-13 Protein Activity is a type of protein that is found inside cells. It was extracted from 3-D cultured constructs and then frozen and pulverized. It was then homogenized in a buffer solution with a protease inhibitor. After that, it was centrifuged and stored at a low temperature. To measure the amount of active MMP-13, a FRET-based assay was used. This assay uses a fluorophore and quencher to measure the amount of MMP-13 that is present. To measure the amount of ECM synthesis, a range of exogenous ATP doses were used. To measure the effect of PPi on MMP-13 activity, chondrocyte monolayer cultures were established and PPi was added to the cultures. To investigate the underlying mechanisms, inhibitors were added to the cultures. Finally, Transmission Electron #Microscopy (TEM) was used to determine the presence of CPPD #crystal accumulation in the engineered tissue constructs. Statistical analyses were then used to analyze the collected data.

The researchers found that when they added ATP to the cultures, MMP-13 activity increased in a dose-dependent manner. This means that the more ATP they added, the more MMP-13 activity increased. They also found that the levels of PPi in the media increased significantly when they added a high dose of ATP, but the levels of PPi in the tissue did not appear to be affected. To determine the best dose of ATP to use, the researchers tested a range of doses and measured the effects on ECM synthesis (collagen and proteoglycans) and MMP-13 activity. They found that ECM synthesis and MMP-13 activity increased in response to intermediate doses of ATP, and further increased in response to higher doses of ATP.

In this study, the researchers wanted to see if they could use ATP to improve tissue growth and mechanical properties without the need for mechanical loading. They found that while high doses of ATP (250 μM) had a positive effect, it also caused a catabolic response, which is when the tissue breaks down. To find the optimal dose of ATP, the researchers tested different doses (31.25, 62.5, and 125 μM) to see which one had the best effect on tissue growth and mechanical properties without causing a catabolic response.

#Calcium is an important factor in the ATP-mediated catabolism process. The researchers found that when they added 10 μM PPi to #chondrocyte cultures, there was a 32% increase in MMP-13 activity compared to unstimulated controls. This effect appeared to require calcium and could be inhibited by the MEK1/2 inhibitor U0126. Additionally, TEM imaging was conducted on engineered cartilaginous tissues supplemented with 0, 62.5 and 250 μM ATP but no mineralization or CPPD crystals were observed which suggests that these doses of ATP did not cause any catabolic response due to crystal formation.

The text is discussing a method of improving tissue growth and mechanical properties of engineered cartilage constructs by applying mechanical loading. However, this approach has limitations when dealing with irregular geometry and high radii of curvature. An alternative approach is to use the known mechanotransduction pathways responsible to achieve the same effect without externally applied forces. In a recent study, it was demonstrated that direct stimulation of the ATP-purinergic receptor pathway through exogenous supplementation of ATP can elicit a comparable anabolic response and be used to improve both tissue growth and mechanical properties of the developed tissue. However, high doses of ATP (250 μM) resulted in a simultaneous catabolic response characterized by an increase in MMP-13 expression, potentially due to the accumulation of ePPi. The present study determined a therapeutic dose range of exogenous ATP to maximize the anabolic response and minimize the catabolic effect of exogenous ATP. It was found that the dose range of ATP between 62.5 and 125 μM was optimal for maximizing the anabolic effect and minimizing the catabolic effect of exogenous ATP. It was also found that calcium and pyrophosphate were key factors involved in the PPi-mediated catabolic response, and that CPPD crystals could potentially be endocytosed and elicit changes through a MAPK-dependent pathway.

#explainpaper #med #MedMastodon

The Therapeutic Potential of Exogenous Adenosine Triphosphate (ATP) for Cartilage Tissue Engineering

authors : Jenna Usprech , Gavin Chu , Renata Giardini-Rosa , Kathleen Martin , and Stephen D. Waldman

It is expected to be a promising agent in the treatment of various ailments and diseases.

Adenosine can be metabolized both externally and internally. Externally, an enzyme called adenosine deaminase is responsible for transforming adenosine into inosine. Additionally, a protein called equilibrative #nucleoside #transporters (ENTs) helps to bring the #extracellular activity of adenosine to an end.

What if we could precisely track a single #virus particle as it is moving around in the #extracellular space at the millisecond timescale?

Researchers at Duke University have developed a fascinating new technique called 3D tracking and #imaging (3D-TrIm).

Research article in Nature Methods: https://www.nature.com/articles/s41592-022-01672-3

Web page of the Welsher lab: https://www.welsherlab.org/3d-trim-captures-viral-first-contacts/

Video source: YouTube

#microscopy #singleparticletracking #infection

Just joined and testing this new platform, time for an #introduction!

We are a #biochemistry and #structuralbiology laboratory based in #Pavia, #Italy.

#wemakeproteins and are fascinated by the #extracellular space: we study how #collagen contributes to the assembly of the #ECM and we perform #drugdiscovery studies on #enzymes of collagen #biosynthesis, plus we investigate various #ligand - #receptor #interactions leading to #signaling in humans and in insects.

Always open to #collaboration.

Check our website and help us grow: http://fornerislab.unipv.it/armenise_lab/