Hypersensitive cell death is known to involve dynamic remodeling of intracellular structures that uses energy released during ATP hydrolysis. However, the relationship between intracellular structural changes and ATP levels during hypersensitive cell death remains unclear. Here, to visualize ATP dynamics directly in real time in individual living plant cells, we applied a genetically encoded Förster resonance energy transfer (FRET)-based fluorescent ATP indicator, ATeam1.03-nD/nA, for plant cells. Intracellular ATP levels increased approximately 3 h after inoculation with the avirulent strain DC3000/avrRpm1 of Pseudomonas syringae pv. tomato (Pst), which was accompanied by the simultaneous disappearance of transvacuolar strands and appearance of bulb-like structures within the vacuolar lumen. Approximately 5 h after bacterial inoculation, the bulb-like structures disappeared and ATP levels drastically decreased. After another 2 h, the large central vacuole was disrupted. In contrast, no apparent changes in intracellular ATP levels were observed in the leaves inoculated with the virulent strain Pst DC3000. The Pst DC3000/avrRpm1-induced hypersensitive cell death was strongly suppressed by inhibiting ATP synthesis after oligomycin A application within 4 h after bacterial inoculation. When the inhibitor was applied 7 h after bacterial inoculation, cell death was unaffected. These observations show that changes in intracellular ATP levels correlate with intracellular morphological changes during hypersensitive cell death, and that ATP is required just before vacuolar rupture in response to bacterial infection.

Pediatric oncology, notably childhood acute lymphoblastic leukemia (ALL), is currently one of the health leading concerns worldwide and a biomedical priority. Decreasing overall leukemia mortality in children requires a comprehensive understanding of its pathobiology. It is becoming clear that malignant cell-to-niche intercommunication and microenvironmental signals that control early cell fate decisions are critical for tumor progression. We show here that the mesenchymal stromal cell component of ALL bone marrow differ from its normal counterpart in a number of functional properties and may have a key role during leukemic development. A decreased proliferation potential, contrasting with the strong ability of producing pro-inflammatory cytokines and an aberrantly loss of CXCL12 and SCF, suggest that leukemic lymphoid niches in ALL bone marrow are unique and may exclude normal hematopoiesis. Cell competence ex vivo assays within tridimensional co-culture structures indicated a growth advantage of leukemic precursor cells and their niche remodeling ability by CXCL12 reduction, resulting in leukemic cell progression at the expense of normal niche-associated lymphopoiesis.

Ca²⁺, Mg²⁺, and K⁺ activities in red beet (Beta vulgaris L.) vacuoles were evaluated using conventional ion-selective microelectrodes and, in the case of Ca²⁺, by non-invasive ion flux measurements (MIFE) as well. The mean vacuolar Ca²⁺ activity was ∼0.2 mM. Modulation of the slow vacuolar (SV) channel voltage dependence by Ca²⁺ in the absence and presence of other cations at their physiological concentrations was studied by patch-clamp in excised tonoplast patches. Lowering pH at the vacuolar side from 7.5 to 5.5 (at zero vacuolar Ca²⁺) did not affect the channel voltage dependence, but abolished sensitivity to luminal Ca²⁺ within a physiological range of concentrations (0.1–1.0 mM). Aggregation of the physiological vacuolar Na⁺ (60 mM) and Mg²⁺ (8 mM) concentrations also results in the SV channel becoming almost insensitive to vacuolar Ca²⁺ variation in a range from nanomoles to 0.1 mM. At physiological cation concentrations at the vacuolar side, cytosolic Ca²⁺ activates the SV channel in a voltage-independent manner with K_d=0.7–1.5 μM. Comparison of the vacuolar Ca²⁺ fluxes measured by both the MIFE technique and from estimating the SV channel activity in attached patches, suggests that, at resting membrane potentials, even at elevated (20 μM) cytosolic Ca²⁺, only 0.5% of SV channels are open. This mediates a Ca²⁺ release of only a few pA per vacuole (∼0.1 pA per single SV channel). Overall, our data suggest that the release of Ca²⁺ through SV channels makes little contribution to a global cytosolic Ca²⁺ signal.

It has been widely described that chronic intake of fructose causes metabolic alterations which can be associated with brain function impairment. In this study, we evaluated the effects of fructose intake on the sleep–wake cycle, locomotion, and neurochemical parameters in Wistar rats. The experimental group was fed with 10% fructose in drinking water for five weeks. After treatment, metabolic indicators were quantified in blood. Electroencephalographic recordings were used to evaluate the sleep architecture and the spectral power of frequency bands. Likewise, the locomotor activity and the concentrations of orexin A and monoamines were estimated. Our results show that fructose diet significantly increased the blood levels of glucose, cholesterol, and triglycerides. Fructose modified the sleep–wake cycle of rats, increasing the waking duration and conversely decreasing the non-rapid eye movement sleep. Furthermore, these effects were accompanied by increases of the spectral power at different frequency bands. Chronic consumption of fructose caused a slight increase in the locomotor activity as well as an increase of orexin A and dopamine levels in the hypothalamus and brainstem. Specifically, immunoreactivity for orexin A was increased in the ventral tegmental area after the intake of fructose. Our study suggests that fructose induces metabolic changes and stimulates the activity of orexinergic and dopaminergic neurons, which may be responsible for alterations of the sleep–wake cycle.

Intracellular $Mg2+$ roles are commensurate with its abundance in the cell cytoplasm. However, little is known about $Mg2+$ subcellular dynamics, primarily due to the lack of suitable $Mg2+$-selective tools to monitor this ion in intracellular compartments. To cope with this lack, we developed a $Mg2+$-sensitive indicator—MagIC (indicator for Magnesium Imaging in Cell) —composed of a functionalized yellow fluorescent protein (FP) variant fused to a red-emitting FP serving as a reference, thus allowing ratiometric imaging of $Mg2+$. MagIC expressed in mammalian cells is homogeneously distributed between the cytosol and nucleus but its fusion with appropriate targeting sequences redirects it to mitochondria or the endoplasmic reticulum. MagIC shows little interference by intracellular $Ca2+$ [$Kd(Mg2+)=5.1mM$; $Kd(Ca2+)=4.8mM$] and its kinetic properties ($koff=84s−1$) approach those of indicator dyes. With MagIC, as reported previously, we also observed a cytosolic $Mg2+$ increase provoked by application of 50 mM $MgCl2$ in the medium. This effect is, however, mimicked by 75 mM KCl or 150 mM d-sorbitol addition, indicating that it is a response to the associated hyperosmotic shock and not to $Mg2+$ itself. Our results confirm the functionality of MagIC as a useful tool for the long-awaited possibility of prolonged and organelle-specific monitoring of cellular $Mg2+$.