Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Arene–Metal π‑Complexation as a Traceless Reactivity Enhancer for C–H Arylation

Current approaches to facilitate C–H arylation of arenes involve the use of either strongly electron-withdrawing substituents or directing groups. Both approaches require structural modification of the arene, limiting their generality. We present a new approach where C–H arylation is made possible without altering the connectivity of the arene via π-complexation of a Cr­(CO)₃ unit, greatly enhancing the reactivity of the aromatic C–H bonds. We apply this approach to monofluorobenzenes, highly unreactive arenes, which upon complexation become nearly as reactive as pentafluorobenzene itself in their couplings with iodoarenes. DFT calculations indicate that C–H activation via a concerted metalation–deprotonation transition state is facilitated by the predisposition of C–H bonds in (Ar–H)­Cr­(CO)₃ to bend out of the aromatic plane

²⁵Mg Solid-State NMR of Magnesium Phosphates: High Magnetic Field Experiments and Density Functional Theory Calculations

Natural-abundance ²⁵Mg solid-state NMR data obtained using very high magnetic fields of 17.6, 20.0, and 30.0 T are reported for a series of magnesium phosphate compounds, some of which are of potential biomedical interest. The ²⁵Mg NMR parameters have been calculated by using the DFT PAW and GIPAW methods, for both the experimental and DFT atomic position optimized structures. For most of the studied compounds, the geometry optimization step improves significantly the accuracy of the calculations and good correlations between experimental and calculated ²⁵Mg chemical shifts and quadrupolar coupling constants were achieved showing that this approach can be used to obtain unambiguous assignments of the ²⁵Mg resonances in more complex phosphate compounds. The possibility of recording natural abundance ²⁵Mg NMR spectra in materials with very low Mg content is illustrated for a ∼10% Mg-substituted hydroxyapatite sample. In this case, the distribution of ²⁵Mg quadrupolar coupling measured experimentally has been compared with values previously calculated for several structural models. The results suggest that more complex structural models must be developed to improve the understanding of the Ca/Mg substitution on the basis of ²⁵Mg NMR data

Convergent (De)Hydrogenative Pathways via a Rhodium α‑Hydroxylalkyl Complex

We report the convergent reaction pathways between [RhH­(PPh₃)₄] and POP ketone (<b>1</b>) and alcohol (<b>2</b>) ligands that terminate in the formation of an α-hydroxylalkyl rhodium­(I) complex (<b>3</b>), representing two halves of a formal reduction/oxidation pathway between <b>1</b> and <b>2</b>. In the case of hydride transfer to <b>1</b>, the formation of the α-hydroxylalkyl rhodium­(I) complex (<b>3</b>) proceeds via a rare hydrido­(η²-carbonyl) complex (<b>4</b>). C–H activation in <b>2</b> at the proligand’s central methine position, rather than O–H activation of the hydroxy motif, followed by loss of dihydrogen also generates the α-hydroxylalkyl rhodium­(I) complex (<b>3</b>). The validity of the postulated reaction pathways is probed with DFT calculations. The observed reactivity supports α-hydroxylalkyl complexes as competent intermediates in ketone hydrogenation catalyzed by rhodium hydrides and suggest that ligands <b>1</b> and <b>2</b> may be “noninnocent” coligands in reported hydrogenation catalyst systems in which they are utilized

Pulsed Electron Paramagnetic Resonance Spectroscopy of ³³S‑Labeled Molybdenum Cofactor in Catalytically Active Bioengineered Sulfite Oxidase

Molybdenum enzymes contain at least one pyranopterin dithiolate (molybdopterin, MPT) moiety that coordinates Mo through two dithiolate (dithiolene) sulfur atoms. For sulfite oxidase (SO), hyperfine interactions (<i>hfi</i>) and nuclear quadrupole interactions (<i>nqi</i>) of magnetic nuclei (<i>I</i> ≠ 0) near the Mo­(V) (d¹) center have been measured using high-resolution pulsed electron paramagnetic resonance (EPR) methods and interpreted with the help of density functional theory (DFT) calculations. These have provided important insights about the active site structure and the reaction mechanism of the enzyme. However, it has not been possible to use EPR to probe the dithiolene sulfurs directly since naturally abundant ³²S has no nuclear spin (<i>I</i> = 0). Here we describe direct incorporation of ³³S (<i>I</i> = 3/2), the only stable magnetic sulfur isotope, into MPT using controlled <i>in vitro</i> synthesis with purified proteins. The electron spin echo envelope modulation (ESEEM) spectra from ³³S-labeled MPT in this catalytically active SO variant are dominated by the “interdoublet” transition arising from the strong nuclear quadrupole interaction, as also occurs for the ³³S-labeled exchangeable equatorial sulfite ligand [Klein, E. L., et al. Inorg. Chem. 2012, 51, 1408−1418]. The estimated experimental <i>hfi</i> and <i>nqi</i> parameters for ³³S (<i>a</i>_iso = 3 MHz and <i>e</i>²<i>Qq</i>/<i>h</i> = 25 MHz) are in good agreement with those predicted by DFT. In addition, the DFT calculations show that the two ³³S atoms are indistinguishable by EPR and reveal a strong intermixing between their out-of-plane p_<i>z</i> orbitals and the d_<i>xy</i> orbital of Mo­(V)

Laboratory Setup for Scanning-Free Grazing Emission X‑ray Fluorescence

Grazing incidence and grazing emission X-ray fluorescence spectroscopy (GI/GE-XRF) are techniques that enable nondestructive, quantitative analysis of elemental depth profiles with a resolution in the nanometer regime. A laboratory setup for soft X-ray GEXRF measurements is presented. Reasonable measurement times could be achieved by combining a highly brilliant laser produced plasma (LPP) source with a scanning-free GEXRF setup, providing a large solid angle of detection. The detector, a pnCCD, was operated in a single photon counting mode in order to utilize its energy dispersive properties. GEXRF profiles of the Ni–L_α,β line of a nickel–carbon multilayer sample, which displays a lateral (bi)­layer thickness gradient, were recorded at several positions. Simulations of theoretical profiles predicted a prominent intensity minimum at grazing emission angles between 5° and 12°, depending strongly on the bilayer thickness of the sample. This information was used to retrieve the bilayer thickness gradient. The results are in good agreement with values obtained by X-ray reflectometry, conventional X-ray fluorescence and transmission electron microscopy measurements and serve as proof-of-principle for the realized GEXRF setup. The presented work demonstrates the potential of nanometer resolved elemental depth profiling in the soft X-ray range with a laboratory source, opening, for example, the possibility of in-line or even in situ process control in semiconductor industry

Analyses of Synthetic <i>N</i>‑Acyl Dopamine Derivatives Revealing Different Structural Requirements for Their Anti-inflammatory and Transient-Receptor-Potential-Channel-of-the-Vanilloid-Receptor-Subfamily-Subtype‑1 (TRPV1)-Activating Properties

We studied the chemical entities within <i>N</i>-octanoyl dopamine (NOD) responsible for the activation of transient-receptor-potential channels of the vanilloid-receptor subtype 1 (TRPV1) and inhibition of inflammation. The potency of NOD in activating TRPV1 was significantly higher compared with those of variants in which the ortho-dihydroxy groups were acetylated, one of the hydroxy groups was omitted (<i>N</i>-octanoyl tyramine), or the ester functionality consisted of a bulky fatty acid (<i>N</i>-pivaloyl dopamine). Shortening of the amide linker (ΔNOD) slightly increased its potency, which was further increased when the carbonyl and amide groups (ΔNODR) were interchanged. With the exception of ΔNOD, the presence of an intact catechol structure was obligatory for the inhibition of VCAM-1 and the induction of HO-1 expression. Because TRPV1 activation and the inhibition of inflammation by <i>N</i>-acyl dopamines require different structural entities, our findings provide a framework for the rational design of TRPV1 agonists with improved anti-inflammatory properties

Analyses of Synthetic <i>N</i>‑Acyl Dopamine Derivatives Revealing Different Structural Requirements for Their Anti-inflammatory and Transient-Receptor-Potential-Channel-of-the-Vanilloid-Receptor-Subfamily-Subtype‑1 (TRPV1)-Activating Properties

We studied the chemical entities within <i>N</i>-octanoyl dopamine (NOD) responsible for the activation of transient-receptor-potential channels of the vanilloid-receptor subtype 1 (TRPV1) and inhibition of inflammation. The potency of NOD in activating TRPV1 was significantly higher compared with those of variants in which the ortho-dihydroxy groups were acetylated, one of the hydroxy groups was omitted (<i>N</i>-octanoyl tyramine), or the ester functionality consisted of a bulky fatty acid (<i>N</i>-pivaloyl dopamine). Shortening of the amide linker (ΔNOD) slightly increased its potency, which was further increased when the carbonyl and amide groups (ΔNODR) were interchanged. With the exception of ΔNOD, the presence of an intact catechol structure was obligatory for the inhibition of VCAM-1 and the induction of HO-1 expression. Because TRPV1 activation and the inhibition of inflammation by <i>N</i>-acyl dopamines require different structural entities, our findings provide a framework for the rational design of TRPV1 agonists with improved anti-inflammatory properties