The recent development of diffraction-unlimited far-field fluorescence microscopy has overcome the classical resolution limit of ~250 nm of conventional light microscopy by about a factor of ten. The improved resolution, however, reveals not only biological structures at an unprecedented resolution, but is also susceptible to sample drift on a much finer scale than previously relevant. Without correction, sample drift leads to smeared images with decreased resolution, and in the worst case to misinterpretation of the imaged structures. This poses a problem especially for techniques such as Fluorescence Photoactivation Localization Microscopy (FPALM/PALM) or Stochastic Optical Reconstruction Microscopy (STORM), which often require minutes recording time. Here we discuss an approach that corrects for three-dimensional (3D) drift in images of fixed samples without the requirement for fiduciary markers or instrument modifications. Drift is determined by calculating the spatial cross-correlation function between subsets of localized particles imaged at different times. Correction down to ~5 nm precision is achieved despite the fact that different molecules are imaged in each frame. We demonstrate the performance of our drift correction algorithm with different simulated structures and analyze its dependence on particle density and localization precision. By imaging mitochondria with Biplane FPALM we show our algorithm’s feasibility in a practical application.

Isocitrate dehydrogenase 2 (IDH2) is located in the mitochondrial matrix. IDH2 acts in the forward Krebs cycle as an NADP⁺-consuming enzyme, providing NADPH for maintenance of the reduced glutathione and peroxiredoxin systems and for self-maintenance by reactivation of cystine-inactivated IDH2 by glutaredoxin 2. In highly respiring cells, the resulting NAD⁺accumulation then induces sirtuin-3-mediated activating IDH2 deacetylation, thus increasing its protective function. Reductive carboxylation of 2-oxoglutarate by IDH2 (in the reverse Krebs cycle direction), which consumes NADPH, may follow glutaminolysis of glutamine to 2-oxoglutarate in cancer cells. When the reverse aconitase reaction and citrate efflux are added, this overall “anoxic” glutaminolysis mode may help highly malignant tumors survive aglycemia during hypoxia. Intermittent glycolysis would hypothetically be required to provide ATP. When oxidative phosphorylation is dormant, this mode causes substantial oxidative stress. Arg172 mutants of human IDH2—frequently found with similar mutants of cytosolic IDH1 in grade 2 and 3 gliomas, secondary glioblastomas, and acute myeloid leukemia—catalyze reductive carboxylation of 2-oxoglutarate and reduction toD-2-hydroxyglutarate, which strengthens the neoplastic phenotype by competitive inhibition of histone demethylation and 5-methylcytosine hydroxylation, leading to genome-wide histone and DNA methylation alternations.D-2-hydroxyglutarate also interferes with proline hydroxylation and thus may stabilize hypoxia-induced factorα.

Significance: Nuclear factor erythroid 2 (NFE2)-related factor 2 ( NFE2L2 , or NRF2) is a transcription factor predominantly affecting the expression of antioxidant genes. NRF2 plays a significant role in the control of redox balance, which is crucial in cancer cells. NRF2 activation regulates numerous cancer hallmarks, including metabolism, cancer stem cell characteristics, tumor aggressiveness, invasion, and metastasis formation. We review the molecular characteristics of the NRF2 pathway and discuss its interactions with the cancer hallmarks previously listed. Recent Advances: The noncanonical activation of NRF2 was recently discovered, and members of this pathway are involved in carcinogenesis. Further, cancer-related changes ( e.g. , metabolic flexibility) that support cancer progression were found to be redox- and NRF2 dependent. Critical Issues: NRF2 undergoes Janus-faced behavior in cancers. The pro- or antineoplastic effects of NRF2 are context dependent and essentially based on the specific molecular characteristics of the cancer in question. Therefore, systematic investigation of NRF2 signaling is necessary to clarify its role in cancer etiology. The biggest challenge in the NRF2 field is to determine which cancers can be targeted for better clinical outcomes. Further, large-scale genomic and transcriptomic studies are missing to correlate the clinical outcome with the activity of the NRF2 system. Future Directions: To exploit NRF2 in a clinical setting in the future, the druggable members of the NRF2 pathway should be identified. In addition, it will be important to study how the modulation of the NRF2 system interferes with cytostatic drugs and their combinations.