Label-Free Characterization of Single Extracellular Vesicles Using Two
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Role of Microbiota-Derived Extracellular Vesicles in Gut-Brain Communication
International Journal of Molecular Sciences Review Role of Microbiota-Derived Extracellular Vesicles in Gut-Brain Communication Carlos M. Cuesta 1, Consuelo Guerri 1 , Juan Ureña 1 and María Pascual 1,2,* 1 Department of Molecular and Cellular Pathology of Alcohol, Príncipe Felipe Research Center, 46012 Valencia, Spain; [email protected] (C.M.C.); [email protected] (C.G.); [email protected] (J.U.) 2 Department of Physiology, School of Medicine and Dentistry, University of Valencia, Avda. Blasco Ibáñez, 15, 46010 Valencia, Spain * Correspondence: [email protected] or [email protected]; Tel.: +34-961-625-635; Fax: +34-963-864-642 Abstract: Human intestinal microbiota comprise of a dynamic population of bacterial species and other microorganisms with the capacity to interact with the rest of the organism and strongly influence the host during homeostasis and disease. Commensal and pathogenic bacteria coexist in homeostasis with the intestinal epithelium and the gastrointestinal tract’s immune system, or GALT (gut-associated lymphoid tissue), of the host. However, a disruption to this homeostasis or dysbiosis by different factors (e.g., stress, diet, use of antibiotics, age, inflammatory processes) can cause brain dysfunction given the communication between the gut and brain. Recently, extracellular vesicles (EVs) derived from bacteria have emerged as possible carriers in gut-brain communication through the interaction of their vesicle components with immune receptors, which lead to neuroinflammatory immune response activation. This review discusses the critical role of bacterial EVs from the gut in the neuropathology of brain dysfunctions by modulating the immune response. These vesicles, which contain harmful bacterial EV contents such as lipopolysaccharide (LPS), peptidoglycans, toxins and Citation: Cuesta, C.M.; Guerri, C.; nucleic acids, are capable of crossing tissue barriers including the blood-brain barrier and interacting Ureña, J.; Pascual, M. -
Quantitative Force Measurements with Optical Tweezers: the JPK Nanotracker™
Quantitative force measurements with optical tweezers: The JPK NanoTracker™ Introduction to optical tweezers manipulate biomolecules and cells, but also to directly and accurately measure the minute forces (on the order of Optical tweezers methodology has developed from proof-of- fractions of picoNewtons) involved. Most often, the principle experiments to an established quantitative biomolecules of interest are not trapped themselves technique in fields ranging from (bio)physics through cell directly, but manipulated through functionalized biology. As the name suggests, optical tweezers are a microspheres. means to manipulate objects with light. With this technique, The correct physical description of optical trapping depends microscopically small objects can be held and manipulated. on the size of the trapped object. One speaks of the ‘ray- At the same time, the forces exerted on the trapped objects optics’ regime when the object’s dimension d is much larger can be accurately measured. The basic physical principle than the wavelength of the trapping light: d>>λ. In this case, underlying optical tweezers is the radiation pressure diffraction effects can be neglected and the trapping forces exerted by light when colliding with matter. For macroscopic of the light can be understood in terms of ray optics. The objects, the radiation pressure exerted by typical light regime where d<<λ is called the Rayleigh regime. In this sources is orders of magnitude too small to have any case, the trapped particles can be treated as point dipoles, measurable effect: we do not feel the light power of the sun as the electromagnetic field is constant on the scale of the pushing us away. -
A Quantum Origin of Life?
June 26, 2008 11:1 World Scientific Book - 9in x 6in quantum Chapter 1 A Quantum Origin of Life? Paul C. W. Davies The origin of life is one of the great unsolved problems of science. In the nineteenth century, many scientists believed that life was some sort of magic matter. The continued use of the term “organic chemistry” is a hangover from that era. The assumption that there is a chemical recipe for life led to the hope that, if only we knew the details, we could mix up the right stuff in a test tube and make life in the lab. Most research on biogenesis has followed that tradition, by assuming chemistry was a bridge—albeit a long one—from matter to life. Elucidat- ing the chemical pathway has been a tantalizing goal, spurred on by the famous Miller-Urey experiment of 1952, in which amino acids were made by sparking electricity through a mixture of water and common gases [Miller (1953)]. But this concept turned out to be something of a blind alley, and further progress with pre-biotic chemical synthesis has been frustratingly slow. In 1944, Erwin Schr¨odinger published his famous lectures under the ti- tle What is Life? [Schr¨odinger (1944)] and ushered in the age of molecular biology. Sch¨odinger argued that the stable transmission of genetic infor- mation from generation to generation in discrete bits implied a quantum mechanical process, although he was unaware of the role of or the specifics of genetic encoding. The other founders of quantum mechanics, including Niels Bohr, Werner Heisenberg and Eugene Wigner shared Schr¨odinger’s belief that quantum physics was the key to understanding the phenomenon Received May 9, 2007 3 QUANTUM ASPECTS OF LIFE © Imperial College Press http://www.worldscibooks.com/physics/p581.html June 26, 2008 11:1 World Scientific Book - 9in x 6in quantum 4 Quantum Aspects of Life of life. -
Depth-Resolved Measurement of Optical Radiation-Pressure Forces with Optical Coherence Tomography
Vol. 26, No. 3 | 5 Feb 2018 | OPTICS EXPRESS 2410 Depth-resolved measurement of optical radiation-pressure forces with optical coherence tomography NICHALUK LEARTPRAPUN, RISHYASHRING R. IYER, AND STEVEN G. ADIE* Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853, USA *[email protected] Abstract: A weakly focused laser beam can exert sufficient radiation pressure to manipulate microscopic particles over a large depth range. However, depth-resolved continuous measurement of radiation-pressure force profiles over an extended range about the focal plane has not been demonstrated despite decades of research on optical manipulation. Here, we present a method for continuous measurement of axial radiation-pressure forces from a weakly focused beam on polystyrene micro-beads suspended in viscous fluids over a depth range of 400 μm, based on real-time monitoring of particle dynamics using optical coherence tomography (OCT). Measurements of radiation-pressure forces as a function of beam power, wavelength, bead size, and refractive index are consistent with theoretical trends. However, our continuous measurements also reveal localized depth-dependent features in the radiation- pressure force profiles that deviate from theoretical predictions based on an aberration-free Gaussian beam. The combination of long-range radiation pressure and OCT offers a new mode of quantitative optical manipulation and detection with extended spatial coverage. This may find applications in the characterization of optical tractor beams, or volumetric optical manipulation and interrogation of beads in viscoelastic media. © 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement OCIS codes: (110.4500) Optical coherence tomography; (350.4855) Optical tweezers or optical manipulation. -
Extracellular Vesicle Proteomes Reflect Developmental Phases of Bacillus Subtilis Yeji Kim1, Nathan Edwards2 and Catherine Fenselau1*
Kim et al. Clin Proteom (2016) 13:6 DOI 10.1186/s12014-016-9107-z Clinical Proteomics RESEARCH Open Access Extracellular vesicle proteomes reflect developmental phases of Bacillus subtilis Yeji Kim1, Nathan Edwards2 and Catherine Fenselau1* Abstract Background: Extracellular vesicles (EV) are spherical membrane-bound vesicles with nano-scale diameters, which are shed to the extracellular region by most eukaryotic and prokaryotic cells. Bacterial EV are proposed to contribute to intercellular communication, bacterial survival and human pathogenesis as a novel secretion system. EV have been characterized from many Gram-negative species and, more recently, from several vegetative Gram-positive bacteria. Further characterization of EV and their molecular cargos will contribute to understanding bacterial physiology and to developing therapeutic approaches. Results: Bacillus subtilis were observed to release EV to a similar extent during sporulation as during the vegetative growth phase. However, the two vesicular cargos show qualitatively and quantitatively different proteomes. Among 193 total proteins identified across both samples, 61 were shown to be significantly more abundant in EV shed by sporulating cells, with (log) ratio of spectral counts RSC > 1 and Fisher-exact test FDR < 5 %. Sixty-two proteins were found to be significantly more abundant in EV shed by vegetative cells. Membrane fusion was shown to take place between these EVs and Gram-positive cells. Conclusion: Biogenesis of EV is a continuous process over the entire life cycle of this sporulating bacterium. The formation of EV during sporulation is strongly supported by the delineation of protein content that differs from the proteome of EV formed by vegetative spores. Keywords: Label-free quantification, Bacillus subtilis, Secretome, Sporulation, Extracellular vesicles Background Gram-positive bacteria were thought not to produce EV Extracellular vesicles (EV) are universally produced because they lack outer membranes. -
Mirna Let-7 from TPO(+) Extracellular Vesicles Is a Potential Marker for a Differential Diagnosis of Follicular Thyroid Nodules
cells Article MiRNA let-7 from TPO(+) Extracellular Vesicles is a Potential Marker for a Differential Diagnosis of Follicular Thyroid Nodules Lidia Zabegina 1,2,3, Inga Nazarova 1,2, Margarita Knyazeva 1,2,3, Nadezhda Nikiforova 1,2, Maria Slyusarenko 1,2, Sergey Titov 4 , Dmitry Vasilyev 1, Ilya Sleptsov 5 and Anastasia Malek 1,2,* 1 Subcellular Technology Lab., N.N. Petrov National Medical Research Center of Oncology, 195251 St. Petersburg, Russia; [email protected] (L.Z.); [email protected] (I.N.); [email protected] (M.K.); [email protected] (N.N.); [email protected] (M.S.); [email protected] (D.V.) 2 Oncosystem Ltd., 121205 Moscow, Russia 3 Institute of Biomedical Systems and Biotechnologies, Peter the Great St. Petersburg Polytechnic University, 195251 St. Petersburg, Russia 4 PCR Laboratory; AO Vector-Best, 630117 Novosibirsk, Russia; [email protected] 5 Department of endocrine surgery, Clinic of High Medical Technologies, St. Petersburg State University N.I. Pirogov, 190103 St. Petersburg, Russia; [email protected] * Correspondence: [email protected]; Tel.: +7-960-250-46-80 Received: 19 July 2020; Accepted: 15 August 2020; Published: 18 August 2020 Abstract: Background: The current approaches to distinguish follicular adenomas (FA) and follicular thyroid cancer (FTC) at the pre-operative stage have low predictive value. Liquid biopsy-based analysis of circulating extracellular vesicles (EVs) presents a promising diagnostic method. However, the extreme heterogeneity of plasma EV population hampers the development of new diagnostic tests. We hypothesize that the isolation of EVs with thyroid-specific surface molecules followed by miRNA analysis, may have improved diagnostic potency. -
Optical Micromachines for Biological Studies
micromachines Review Optical Micromachines for Biological Studies Philippa-Kate Andrew 1 , Martin A. K. Williams 2,3 and Ebubekir Avci 1,3,* 1 Department of Mechanical and Electrical Engineering, Massey University, Palmerston North 4410, New Zealand; [email protected] 2 School of Fundamental Sciences, Massey University, Palmerston North 4410, New Zealand; [email protected] 3 MacDiarmid Institute for Advanced Materials and Nanotechnology, Wellington 6140, New Zealand * Correspondence: [email protected] Received: 21 January 2020; Accepted: 9 February 2020; Published: 13 February 2020 Abstract: Optical tweezers have been used for biological studies since shortly after their inception. However, over the years research has suggested that the intense laser light used to create optical traps may damage the specimens being studied. This review aims to provide a brief overview of optical tweezers and the possible mechanisms for damage, and more importantly examines the role of optical micromachines as tools for biological studies. This review covers the achievements to date in the field of optical micromachines: improvements in the ability to produce micromachines, including multi-body microrobots; and design considerations for both optical microrobots and the optical trapping set-up used for controlling them are all discussed. The review focuses especially on the role of micromachines in biological research, and explores some of the potential that the technology has in this area. Keywords: optical tweezers; multi-component micromanipulators; radiation damage; life sciences; optical microrobots 1. Introduction Improvements in tools for the visualisation of objects at the micro- and nano- scale have given researchers the ability to investigate materials and processes previously out of reach. -
Exploring Transcriptomic Landscapes in Red Blood Cells, in Their Extracellular Vesicles and on a Single- Cell Level
Exploring transcriptomic landscapes in red blood cells, in their extracellular vesicles and on a single- cell level Erja Kerkelä ( erja.kerkela@bloodservice. ) Finnish Red Cross Blood Service https://orcid.org/0000-0002-2923-5300 Jenni Lahtela University of Helsinki, Institute for Molecular Medicine Finland (FIMM) Antti Larjo Finnish Red Cross Blood Service Ulla Impola Finnish Red Cross Blood Service Laura Mäenpää University of Helsinki, Research Programs Unit, Molecular Neurology Pirkko Mattila University of Helsinki, Institute of Molecular Medicine Finland (FIMM) Research article Keywords: erythrocyte, MALAT1, RNA sequencing, transcriptomics, extracellular vesicle Posted Date: September 18th, 2019 DOI: https://doi.org/10.21203/rs.2.14503/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/27 Abstract Background Circulating human red blood cells (RBCs) consist of mature erythrocytes and immature reticulocytes. Being anucleated, RBCs lack typical transcriptomes, but are known to contain small amounts of diverse long transcripts and microRNAs. However, the exact role and importance of these RNAs is lacking. Results To study this further, we explored the transcriptomes of RBCs and extracellular vesicles (EVs) of RBCs using next-generation sequencing. Furthermore, to understand the dynamics of the RBC transcriptome, we performed single-cell RNA sequencing on RBCs. An analysis of the single-cell transcriptomes revealed that approximately 10% of the cells contained detectable levels of mRNA and fell into three subpopulations based on their transcriptomes. Decrease in the mRNA quantity was observed across the populations. Qualitative changes included the differences in the globin transcripts and changes in the expression of ribosomal genes. -
Pulsed Optical Tweezers for Levitation and Manipulation of Stuck Biological
2005 Conference on Lasers & Electro-Optics (CLEO) CFN3 Pulsed optical tweezers for levitation and manipulation of stuck biological particles Amol Ashok Ambardekar and Yong-qing Li Department ofPhysics, East Carolina University, Greenville, North Carolina 27858 acube3(1!yahoo. covm,livrnail.ecu. ec/ Abstract: We report on optical levitation and manipulation of microscopic particles that are stuck on a glass surface with a pulsed optical tweezers. Both the stuck dielectric beads and biological cells are demonstrated to be levitated. ©2005 Optical Society of America OCIS codes: (170.4520) Optical confinement and manipulation; (140.7010) Trapping Optical tweezers has become a powerful tool for capturing and manipulation of micron-sized particles, typically by using continuous-wave (cw) lasers [1, 2]. It has been routinely applied to manipulate living cells, bacteria, viruses, chromosomes and other organelles [3, 4], To reduce the photodamage to the trapped particles, the average power of the trapping lasers is usually limited to below hundreds of mW and near-infrared (NIR) or infrared lasers were used for trapping [3, 6]. The trapping force generated by the cw optical tweezers is typically in the order of 10-12 N [4, 5]. This weak force is efficient to confine micro-particles suspended in liquids, but not sufficient to levitate the particles that are stuck on the glass surface, where it has to overcome the binding force. Therefore, the stuck particles cannot be manipulated with the optical tweezers that only employs cw lasers. In this paper, we describe a pulsed optical tweezers that employs a pulsed laser for levitation of the stuck particles and a low-power cw laser for successive trapping and manipulation. -
Extracellular Vesicles in Immunomodulation and Tumor Progression
REVIEW ARTICLE https://doi.org/10.1038/s41590-021-00899-0 Extracellular vesicles in immunomodulation and tumor progression Carolyn Marar1,2,6,7, Bartholomew Starich 1,3,7 and Denis Wirtz 1,3,4,5 ✉ Extracellular vesicles have emerged as prominent regulators of the immune response during tumor progression. EVs contain a diverse repertoire of molecular cargo that plays a critical role in immunomodulation. Here, we identify the role of EVs as media- tors of communication between cancer and immune cells. This expanded role of EVs may shed light on the mechanisms behind tumor progression and provide translational diagnostic and prognostic tools for immunologists. ells communicate via contact-dependent and contact- described the ability of small EVs released by dendritic cells (DCs) independent interactions through the secretion of chemo- to induce tumor suppression in vivo. In 2002, another study elabo- kines, cytokines, growth factors, and associated cell recep- rated on this work to describe a mechanism for indirect T cell stim- C 11 tors. Over the past 30 years, the secretion of EVs has also emerged ulation by DC small EVs . These discoveries established EVs as a as a major mechanism for cell–cell and cell–environment interac- major mechanism of cellular communication and provided a basis tions. These 30- to 5,000-nm lipid-membrane-bound vesicles con- for immune EV research over the past two decades. tain functional proteins, nucleic acids, lipids, and other bioactive DCs are antigen-presenting cells (APCs) that orchestrate the molecules that can modulate the behavior of recipient cells1. immune response against a specific antigen. -
Extracellular Vesicles and Host–Pathogen Interactions: a Review of Inter-Kingdom Signaling by Small Noncoding RNA
G C A T T A C G G C A T genes Review Extracellular Vesicles and Host–Pathogen Interactions: A Review of Inter-Kingdom Signaling by Small Noncoding RNA Bruce A. Stanton Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA; [email protected] Abstract: The focus of this brief review is to describe the role of noncoding regulatory RNAs, including short RNAs (sRNA), transfer RNA (tRNA) fragments and microRNAs (miRNA) secreted in extracellular vesicles (EVs), in inter-kingdom communication between bacteria and mammalian (human) host cells. Bacteria secrete vesicles that contain noncoding regulatory RNAs, and recent studies have shown that the bacterial vesicles fuse with and deliver regulatory RNAs to host cells, and similar to eukaryotic miRNAs, regulatory RNAs modulate the host immune response to infection. Recent studies have also demonstrated that mammalian cells secrete EVs containing miRNAs that regulate the gut microbiome, biofilm formation and the bacterial response to antibiotics. Thus, as evidence accumulates it is becoming clear that the secretion of noncoding regulatory RNAs and miRNAs in extracellular vesicles is an important mechanism of bidirectional communication between bacteria and mammalian (human) host cells. However, additional research is necessary to elucidate how noncoding regulatory RNAs and miRNA secreted in extracellular vesicles mediate inter-kingdom communication. Keywords: outer membrane vesicles (OMV); extracellular vesicles (EVs); bacterial extracellular Citation: Stanton, B.A. Extracellular vesicles (BEV); small non-coding RNA (sRNA); inter-kingdom communication Vesicles and Host–Pathogen Interactions: A Review of Inter-Kingdom Signaling by Small Noncoding RNA. Genes 2021, 12, 1010. -
Radioiodine Labeling and in Vivo Trafficking of Extracellular Vesicles
www.nature.com/scientificreports OPEN Radioiodine labeling and in vivo trafcking of extracellular vesicles Chae Moon Hong1,2, Prakash Gangadaran1,3, Ji Min Oh1, Ramya Lakshmi Rajendran1, Arunnehru Gopal1, Liya Zhu1 & Byeong‑Cheol Ahn1,2* Biodistribution and role of extracellular vesicles (EVs) are still largely unknown. Reliable tracking methods for EVs are needed. In this study, nuclear imaging using radioiodine were developed and applied for tracking EVs derived from cell lines. EVs were obtained from supernatant of thyroid cancer cell (Cal62) and natural killer cells (NK92‑MI) using sequential ultracentrifuges. Sulfosuccinimidyl‑3‑(4‑ hydroxypheynyl) propionate were labeled to membrane of Cal62 and NK92‑MI cell derived EVs, then the EVs were labeled with radioiodine (I‑131 and I‑125) using pre‑coated iodination tubes (RI‑EVs). In vivo gamma camera images were obtained after intravenous injection of the RI‑EVs, and ex vivo biodistribution study was also performed. EVs were labeled with radioiodine and radiochemical purity of the RI‑EV was more than 98%. Results of nanoparticle tracking analysis and electron microscopy showed that there was no signifcant diference in EVs before and after the radioiodine labeling. After intravenous injection of RI‑EVs to mice, gamma camera imaging well visualized the real‑time biodistribution of the RI‑EVs. RI‑EVs were mainly visualized at liver, spleen, and lung. Nuclear imaging system of EVs derived from thyroid cancer and NK cells using radioiodine labeling of the EVs was established. Thus, this system might be helpful for in vivo tracking of EVs. Extracellular vesicles (EVs) are naturally released from the cell that are delimited by a lipid bilayer and cannot replicate, which do not contain functional nucleus1.