Cell-specific modulation of mitochondrial respiration and metabolism by the pro-apoptotic Bcl-2 family members Bax and Bak.

Proteins from the Bcl-2 family play an essential role in the regulation of apoptosis. However, they also possess cell death-unrelated activities that are less well understood. This prompted us to study apoptosis-unrelated activities of the Bax and Bak, pro-apoptotic members of the Bcl-2 family. We prepared Bax/Bak-deficient human cancer cells of different origin and found that while respiration in the glioblastoma U87 Bax/Bak-deficient cells was greatly enhanced, respiration of Bax/Bak-deficient B lymphoma HBL-2 cells was slightly suppressed. Bax/Bak-deficient U87 cells also proliferated faster in culture, formed tumours more rapidly in mice, and showed modulation of metabolism with a considerably increased NAD+/NADH ratio. Follow-up analyses documented increased/decreased expression of mitochondria-encoded subunits of respiratory complexes and stabilization/destabilization of the mitochondrial transcription elongation factor TEFM in Bax/Bak-deficient U87 and HBL-2 cells, respectively. TEFM downregulation using shRNAs attenuated mitochondrial respiration in Bax/Bak-deficient U87 as well as in parental HBL-2 cells. We propose that (post)translational regulation of TEFM levels in Bax/Bak-deficient cells modulates levels of subunits of mitochondrial respiratory complexes that, in turn, contribute to respiration and the accompanying changes in metabolism and proliferation in these cells.

Metastable Brominated Nanodiamond Surface Enables Room Temperature and Catalysis-Free Amine Chemistry.

Bromination of high-pressure, high-temperature (HPHT) nanodiamond (ND) surfaces has not been explored and can open new avenues for increased chemical reactivity and diamond lattice covalent bond formation. The large bond dissociation energy of the diamond lattice-oxygen bond is a challenge that prevents new bonds from forming, and most researchers simply use oxygen-terminated NDs (alcohols and acids) as reactive species. In this work, we transformed a tertiary-alcohol-rich ND surface to an amine surface with ∼50% surface coverage and was limited by the initial rate of bromination. We observed that alkyl bromide moieties are highly labile on HPHT NDs and are metastable as previously found using density functional theory. The strong leaving group properties of the alkyl bromide intermediate were found to form diamond-nitrogen bonds at room temperature and without catalysts. This robust pathway to activate a chemically inert ND surface broadens the modalities for surface termination, and the unique surface properties of brominated and aminated NDs are impactful to researchers for chemically tuning diamond for quantum sensing or biolabeling applications.

Francisella tularensis Outer Membrane Vesicles Participate in the Early Phase of Interaction With Macrophages.

Francisella tularensis is known to release unusually shaped tubular outer membrane vesicles (OMV) containing a number of previously identified virulence factors and immunomodulatory proteins. In this study, we present that OMV isolated from the F. tularensis subsp. holarctica strain FSC200 enter readily into primary bone marrow-derived macrophages (BMDM) and seem to reside in structures resembling late endosomes in the later intervals. The isolated OMV enter BMDM generally via macropinocytosis and clathrin-dependent endocytosis, with a minor role played by lipid raft-dependent endocytosis. OMVs proved to be non-toxic and had no negative impact on the viability of BMDM. Unlike the parent bacterium itself, isolated OMV induced massive and dose-dependent proinflammatory responses in BMDM. Using transmission electron microscopy, we also evaluated OMV release from the bacterial surface during several stages of the interaction of Francisella with BMDM. During adherence and the early phase of the uptake of bacteria, we observed numerous tubular OMV-like protrusions bulging from the bacteria in close proximity to the macrophage plasma membrane. This suggests a possible role of OMV in the entry of bacteria into host cells. On the contrary, the OMV release from the bacterial surface during its cytosolic phase was negligible. We propose that OMV play some role in the extracellular phase of the interaction of Francisella with the host and that they are involved in the entry mechanism of the bacteria into macrophages.

Nanoscale Dynamic Readout of a Chemical Redox Process Using Radicals Coupled with Nitrogen-Vacancy Centers in Nanodiamonds.

Biocompatible nanoscale probes for sensitive detection of paramagnetic species and molecules associated with their (bio)chemical transformations would provide a desirable tool for a better understanding of cellular redox processes. Here, we describe an analytical tool based on quantum sensing techniques. We magnetically coupled negatively charged nitrogen-vacancy (NV) centers in nanodiamonds (NDs) with nitroxide radicals present in a bioinert polymer coating of the NDs. We demonstrated that the T1 spin relaxation time of the NV centers is very sensitive to the number of nitroxide radicals, with a resolution down to ∼10 spins per ND (detection of approximately 10-23 mol in a localized volume). The detection is based on T1 shortening upon the radical attachment, and we propose a theoretical model describing this phenomenon. We further show that this colloidally stable, water-soluble system can be used dynamically for spatiotemporal readout of a redox chemical process (oxidation of ascorbic acid) occurring near the ND surface in an aqueous environment under ambient conditions.

Simultaneous label-free live imaging of cell nucleus and luminescent nanodiamonds.

In recent years, fluorescent nanodiamond (fND) particles containing nitrogen-vacancy (NV) centers gained recognition as an attractive probe for nanoscale cellular imaging and quantum sensing. For these applications, precise localization of fNDs inside of a living cell is essential. Here we propose such a method by simultaneous detection of the signal from the NV centers and the spectroscopic Raman signal from the cells to visualize the nucleus of living cells. However, we show that the commonly used Raman cell signal from the fingerprint region is not suitable for organelle imaging in this case. Therefore, we develop a method for nucleus visualization exploiting the region-specific shape of C-H stretching mode and further use k-means cluster analysis to chemically distinguish the vicinity of fNDs. Our technique enables, within a single scan, to detect fNDs, distinguish by chemical localization whether they have been internalized into cell and simultaneously visualize cell nucleus without any labeling or cell-fixation. We show for the first time spectral colocalization of unmodified high-pressure high-temperature fND probes with the cell nucleus. Our methodology can be, in principle, extended to any red- and near-infrared-luminescent cell-probes and is fully compatible with quantum sensing measurements in living cells.

Formation of gadolinium-ferritin from clinical magnetic resonance contrast agents.

Gadolinium deposition in the brain following administration of gadolinium-based contrast agents (GBCAs) has led to health concerns. We show that some clinical GBCAs form Gd3+-ferritin nanoparticles at (sub)nanomolar concentrations of Gd3+ under physiological conditions. We describe their structure at atomic resolution and discuss potential relevance for clinical MRI.

Diamond nano-optode for fluorescent measurements of pH and temperature.

Nano-optodes with a diamond core coated with a double stimuli-responsive polymeric shell reversibly respond to pH and temperature changes. Swelling and collapsing of the shell are accompanied by changes in the charge of the polymer. Changes in the fluorescent spectra of nitrogen-vacancy centers ratiometrically indicate pH and temperature.

Extremely rapid isotropic irradiation of nanoparticles with ions generated in situ by a nuclear reaction.

Energetic ions represent an important tool for the creation of controlled structural defects in solid nanomaterials. However, the current preparative irradiation techniques in accelerators show significant limitations in scaling-up, because only very thin layers of nanoparticles can be efficiently and homogeneously irradiated. Here, we show an easily scalable method for rapid irradiation of nanomaterials by light ions formed homogeneously in situ by a nuclear reaction. The target nanoparticles are embedded in B2O3 and placed in a neutron flux. Neutrons captured by 10B generate an isotropic flux of energetic α particles and 7Li+ ions that uniformly irradiates the surrounding nanoparticles. We produced 70 g of fluorescent nanodiamonds in an approximately 30-minute irradiation session, as well as fluorescent silicon carbide nanoparticles. Our method thus increased current preparative yields by a factor of 102-103. We envision that our technique will increase the production of ion-irradiated nanoparticles, facilitating their use in various applications.

Anchored but not internalized: shape dependent endocytosis of nanodiamond.

Nanoparticle-cell interactions begin with the cellular uptake of the nanoparticles, a process that eventually determines their cellular fate. In the present work, we show that the morphological features of nanodiamonds (NDs) affect both the anchoring and internalization stages of their endocytosis. While a prickly ND (with sharp edges/corners) has no trouble of anchoring onto the plasma membrane, it suffers from difficult internalization afterwards. In comparison, the internalization of a round ND (obtained by selective etching of the prickly ND) is not limited by its lower anchoring amount and presents a much higher endocytosis amount. Molecular dynamics simulation and continuum modelling results suggest that the observed difference in the anchoring of round and prickly NDs likely results from the reduced contact surface area with the cell membrane of the former, while the energy penalty associated with membrane curvature generation, which is lower for a round ND, may explain its higher probability of the subsequent internalization.