Transcriptome of Extracellular Vesicles: State-of-the-Art

Exosomes and microvesicles are two major categories of extracellular vesicles (EVs) released by almost all cell types and are highly abundant in biological fluids. Both the molecular composition of EVs and their release are thought to be strictly regulated by external stimuli. Multiple studies have consistently demonstrated that EVs transfer proteins, lipids and RNA between various cell types, thus mediating intercellular communication, and signaling. Importantly, small non-coding RNAs within EVs are thought to be major contributors to the molecular events occurring in the recipient cell. Furthermore, RNA cargo in exosomes and microvesicles could hold tremendous potential as non-invasive biomarkers for multiple disorders, including pathologies of the immune system. This mini-review is aimed to provide the state-of-the-art in the EVs-associated RNA transcriptome field, as well as the comprehensive analysis of previous studies characterizing RNA content within EVs released by various cells using next-generation sequencing. Finally, we highlight the technical challenges associated with obtaining pure EVs and deep sequencing of the EV-associated RNAs

Elevated intracellular chloride level in albino visual cortex neurons is mediated by Na-K-Cl co-transporter

<p>Abstract</p> <p>Background</p> <p>During development the switch from a depolarizing to a hyperpolarizing action of GABA is a consequence of a decrease of the Na⁺-K⁺-2Cl^-co-transporter (NKCC1, Cl^--uptake) and increase of the K⁺-Cl^-co-transporter (KCC2, Cl^--extrusion) expression. However albino visual cortex neurons don't show a corresponding decrease in intracellular chloride concentration during development of the visual system as compared to pigmented animals.</p> <p>Results</p> <p>Our study revealed that more cells express NKCC1 in albinos compared to pigmented rat visual cortex neurons whereas KCC2 is expressed in all cells in both strains. We determined a positive relationship between the presence of NKCC1 and an inhibitory deficit in single neurons of the albino visual cortex. After pharmacological blockade of NKCC1 function with its specific inhibitor, bumetanide, the reversal potential of electrically evoked GABAA receptor-mediated postsynaptic currents and, as a consequence, [Cl-]_iin albino visual cortex neurons shifted to the pigmented rat brain value. In conclusion, our pharmacological experiments and subsequent single cell real time PCR analysis of the co-transporter mRNA demonstrated that the inhibitory deficit present in the albino visual cortical network is almost exclusively mediated by NKCC1.</p> <p>Conclusion</p> <p>Our findings suggest that blocking of NKCC1 in albino visual cortex neurons could improve processing in visual cortex and therefore might be beneficial for vision in albinos.</p

The 2023 terahertz science and technology roadmap

Terahertz (THz) radiation encompasses a wide spectral range within the electromagnetic spectrum that extends from microwaves to the far infrared (100 GHz–∼30 THz). Within its frequency boundaries exist a broad variety of scientific disciplines that have presented, and continue to present, technical challenges to researchers. During the past 50 years, for instance, the demands of the scientific community have substantially evolved and with a need for advanced instrumentation to support radio astronomy, Earth observation, weather forecasting, security imaging, telecommunications, non-destructive device testing and much more. Furthermore, applications have required an emergence of technology from the laboratory environment to production-scale supply and in-the-field deployments ranging from harsh ground-based locations to deep space. In addressing these requirements, the research and development community has advanced related technology and bridged the transition between electronics and photonics that high frequency operation demands. The multidisciplinary nature of THz work was our stimulus for creating the 2017 THz Science and Technology Roadmap (Dhillon et al 2017 J. Phys. D: Appl. Phys. 50 043001). As one might envisage, though, there remains much to explore both scientifically and technically and the field has continued to develop and expand rapidly. It is timely, therefore, to revise our previous roadmap and in this 2023 version we both provide an update on key developments in established technical areas that have important scientific and public benefit, and highlight new and emerging areas that show particular promise. The developments that we describe thus span from fundamental scientific research, such as THz astronomy and the emergent area of THz quantum optics, to highly applied and commercially and societally impactful subjects that include 6G THz communications, medical imaging, and climate monitoring and prediction. Our Roadmap vision draws upon the expertise and perspective of multiple international specialists that together provide an overview of past developments and the likely challenges facing the field of THz science and technology in future decades. The document is written in a form that is accessible to policy makers who wish to gain an overview of the current state of the THz art, and for the non-specialist and curious who wish to understand available technology and challenges. A such, our experts deliver a 'snapshot' introduction to the current status of the field and provide suggestions for exciting future technical development directions. Ultimately, we intend the Roadmap to portray the advantages and benefits of the THz domain and to stimulate further exploration of the field in support of scientific research and commercial realisation

Characterization of extracellular circulating microRNA

MicroRNAs (miRNAs), a class of post-transcriptional gene expression regulators, have recently been detected in human body fluids, including peripheral blood plasma as extracellular nuclease resistant entities. However, the origin and function of extracellular circulating miRNA remain essentially unknown. Here, we confirmed that circulating mature miRNA in contrast to mRNA or snRNA is strikingly stable in blood plasma and cell culture media. Furthermore, we found that most miRNA in plasma and cell culture media completely passed through 0.22 µm filters but remained in the supernatant after ultracentrifugation at 110 000g indicating the non-vesicular origin of the extracellular miRNA. Furthermore, western blot immunoassay revealed that extracellular miRNA ultrafiltrated together with the 96 kDa Ago2 protein, a part of RNA-induced silencing complex. Moreover, miRNAs in both blood plasma and cell culture media co-immunoprecipited with anti-Ago2 antibody in a detergent free environment. This is the first study to show that extracellular miRNAs are predominantly exosomes/microvesicles free and are associated with Ago proteins. We hypothesize that extracellular miRNAs are in the most part by-products of dead cells that remain in extracellular space due to the high stability of the Ago2 protein and Ago2-miRNA complex. Nevertheless, our data does not reject the possibility that some miRNAs can be associated with exosomes

Relationships of APOE genotypes with small RNA and protein cargo of brain tissue extracellular vesicles from patients with late-stage AD

Background and Objectives Variants of the apolipoprotein E (APOE) gene are the greatest known risk factors for sporadic Alzheimer disease (AD). Three major APOE isoform alleles, ϵ2, ϵ3, and ϵ4, encode and produce proteins that differ by only 1-2 amino acids but have different binding partner interactions. Whereas APOE ϵ2 is protective against AD relative to ϵ3, ϵ4 is associated with an increased risk for AD development. However, the role of APOE in gene regulation in AD pathogenesis has remained largely undetermined. Extracellular vesicles (EVs) are lipid bilayer-delimited particles released by cells to dispose of unwanted materials and mediate intercellular communication, and they are implicated in AD pathophysiology. Brain-derived EVs (bdEVs) could act locally in the tissue and reflect cellular changes. To reveal whether APOE genotype affects EV components in AD brains, bdEVs were separated from patients with AD with different APOE genotypes for parallel small RNA and protein profile. Methods bdEVs from late-stage AD brains (BRAAK stages 5-6) from patients with APOE genotypes ϵ2/3 (n = 5), ϵ3/3 (n = 5), ϵ3/4 (n = 6), and ϵ4/4 (n = 6) were separated using our published protocol into a 10,000g pelleted extracellular fraction (10K) and a further purified EV fraction. Counting, sizing, and multiomic characterization by small RNA sequencing and proteomic analysis were performed for 10K, EVs, and source tissue. Results Comparing APOE genotypes, no significant differences in bdEV total particle concentration or morphology were observed. Overall small RNA and protein profiles of 10K, EVs, and source tissue also did not differ substantially between different APOE genotypes. However, several differences in individual RNAs (including miRNAs and tRNAs) and proteins in 10K and EVs were observed when comparing the highest and lowest risk groups (ϵ4/4 and ϵ2/3). Bioinformatic analysis and previous publications indicate a potential regulatory role of these molecules in AD. Discussion For patients with late-stage AD in this study, only a few moderate differences were observed for small RNA and protein profiles between APOE genotypes. Among these, several newly identified 10K and EV-associated molecules may play roles in AD progression. Possibly, larger genotype-related differences exist and are more apparent in or before earlier disease stages

Functional studies of gap junction protein connexin43 using RNA interference in vitro and in vivo

In dieser Arbeit wurde eine auf RNA-Interferenz (RNAi) basierende Methode für die funktionelle Charakterisierung des $\textit {gap junction}$ Proteins Connexin43 (Cx43) entwickelt. Das Hauptinteresse galt der Frage, ob Cx43 eine Zielstruktur im Prozess der glialen Narbenbildung ($\textit {glial scarring}$) darstellt. Wir haben herausgefunden, dass die RNAi vermittelte Herabregulation von Cx43 in kultivierten Astrocyten deren Proliferationsrate verringert. Zudem zeigten mit siRNA behandelte Astrocyten eine verstärkte Migration und geringere Adhäsion $\textit {in vitro}$. Daneben wurde eine Reihe von Bedingungen getestet, um siRNA gezielt in der Mausretina zu applizieren. Das optimierte Protokoll, in dem die siRNA mit dem Transit-TKO Transfektionsreagenz komplexiert wurde, sorgt für eine Diffusion der Komplexe bis zur Ganglien-Zellschicht der Retina, einschließlich astrocytärer Netzwerke. Das etablierte Protokoll schafft die Grundlage für $\textit {in vivo}$ Studien an der Mausretina, die ein Modell für $\textit {glial scarring}$ ist.In this work we explored an RNA interference strategy for functional studies of the gap junction protein Cx43. The major interest of the project was the question if the Cx43 can be used as a target for the prevention of glial scarring. We found that acute knockdown of the Cx43 in cultured astrocytes correlated with the decreased proliferation rate of the cells. In addition, Cx43 impaired astrocytes showed significantly enhanced migration and lower adhesion capacity in vitro. Secondly, we have tested a number of strategies to deliver synthetic siRNA into the mouse retina in vivo. We had shown that under specific protocol siRNA conjugated with Transit-TKO transfection reagent was able to penetrate into the ganglion cell layer of mouse retina. Moreover, siRNA was localized within the retinal astrocytes network. The ultimate goal would be to study the functional effect of the siRNA mediated Cx43 downregulation on glial scar formation in a retina model of neurotrauma

The origin, function and diagnostic potential of extracellular microRNA in human body fluids

Short non-coding RNA molecules, microRNAs (miRNAs), post-transcriptionally regulate gene expression in living cells. In recent years, miRNAs have been found in a wide spectrum of mammalian body fluids including blood plasma, saliva, urine, milk, seminal plasma, tears and amniotic fluid as extracellular circulating nuclease-resistant entities. The changes in miRNA spectra observed in certain fluids correlated with various pathological conditions suggesting that extracellular miRNAs can serve as informative biomarkers for certain diseases including cancer. However, the mechanism of generation and a biological role of extracellular miRNAs remain unclear. The current theories regarding extracellular miRNA origin and function suggest that these miRNAs can be either non-specific 'by-products' of cellular activity and cell death or specifically released cell-cell signaling messengers. The goal of this Research Topic is to bring together up-to-date knowledge about the extracellular miRNA and its role in disease diagnostics and, possibly, inter-cellular communication