24 research outputs found
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)<sub>3</sub> 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)<sub>3</sub> to bend out of the aromatic
plane
Weakly Nonlinear Analysis of Vortex Formation in a Dissipative Variant of the Gross--Pitaevskii Equation
<sup>25</sup>Mg Solid-State NMR of Magnesium Phosphates: High Magnetic Field Experiments and Density Functional Theory Calculations
Natural-abundance <sup>25</sup>Mg 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 <sup>25</sup>Mg 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 <sup>25</sup>Mg chemical shifts
and quadrupolar coupling constants were achieved showing that this
approach can be used to obtain unambiguous assignments of the <sup>25</sup>Mg resonances in more complex phosphate compounds. The possibility
of recording natural abundance <sup>25</sup>Mg NMR spectra in materials
with very low Mg content is illustrated for a ∼10% Mg-substituted
hydroxyapatite sample. In this case, the distribution of <sup>25</sup>Mg 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 <sup>25</sup>Mg NMR data
Convergent (De)Hydrogenative Pathways via a Rhodium α‑Hydroxylalkyl Complex
We
report the convergent reaction pathways between [RhH(PPh<sub>3</sub>)<sub>4</sub>] 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(η<sup>2</sup>-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 <sup>33</sup>S‑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) (d<sup>1</sup>) 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 <sup>32</sup>S has no nuclear spin (<i>I</i> =
0). Here we describe direct incorporation of <sup>33</sup>S (<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 <sup>33</sup>S-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 <sup>33</sup>S-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 <sup>33</sup>S (<i>a</i><sub>iso</sub> = 3 MHz and <i>e</i><sup>2</sup><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 <sup>33</sup>S atoms are indistinguishable
by EPR and reveal a strong intermixing between their out-of-plane
p<sub><i>z</i></sub> orbitals and the d<sub><i>xy</i></sub> 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<sub>α,β</sub> 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