Targeting PKCα alleviates iron overload in diabetes and hemochromatosis through the inhibition of ferroportin.

ABSTRACT: Ferroportin (Fpn) is the only iron exporter, playing a crucial role in systemic iron homeostasis. Fpn is negatively regulated by its ligand hepcidin, but other potential regulators in physiological and disease conditions remain poorly understood. Diabetes is a metabolic disorder that develops body iron loading with unknown mechanisms. By using diabetic mouse models and human duodenal specimens, we demonstrated that intestinal Fpn expression was increased in diabetes in a hepcidin-independent manner. Protein kinase C (PKC) is hyperactivated in diabetes. We showed that PKCα was required to sustain baseline Fpn expression and diabetes-induced Fpn upregulation in the enterocytes and macrophages. Knockout of PKCα abolished diabetes-associated iron overload. Mechanistically, activation of PKCα increased the exocytotic trafficking of Fpn and decreased the endocytic trafficking of Fpn in the resting state. Hyperactive PKCα also suppressed hepcidin-induced ubiquitination, internalization, and degradation of Fpn. We further observed that iron loading in the enterocytes and macrophages activated PKCα, acting as a novel mechanism to enhance Fpn-dependent iron efflux. Finally, we demonstrated that the loss-of-function of PKCα and pharmacological inhibition of PKC significantly alleviated hereditary hemochromatosis-associated iron overload. Our study has highlighted, to our knowledge, for the first time, that PKCα is an important positive regulator of Fpn and a new target in the control of iron homeostasis.

Speckle-correlation imaging through a kaleidoscopic multimode fiber.

Speckle-correlation imaging techniques are widely used for noninvasive imaging through complex scattering media. While light propagation through multimode fibers and scattering media share many analogies, reconstructing images through multimode fibers from speckle correlations remains an unsolved challenge. Here, we exploit a kaleidoscopic memory effect emerging in square-core multimode fibers and demonstrate fluorescence imaging with no prior knowledge on the fiber. Experimentally, our approach simply requires to translate random speckle patterns at the input of a square-core fiber and to measure the resulting fluorescence intensity with a bucket detector. The image of the fluorescent object is then reconstructed from the autocorrelation of the measured signal by solving an inverse problem. This strategy does not require the knowledge of the fragile deterministic relation between input and output fields, which makes it promising for the development of flexible minimally invasive endoscopes.

Shaping an evanescent focus of light for high spatial resolution optogenetic activations in live cells.

Confining light illumination in the three dimensions of space is a challenge for various applications. Among these, optogenetic methods developed for live experiments in cell biology would benefit from such a localized illumination as it would improve the spatial resolution of diffusive photosensitive proteins leading to spatially constrained biological responses in specific subcellular organelles. Here, we describe a method to create and move a focused evanescent spot, at the interface between a glass substrate and an aqueous sample, across the field of view of a high numerical aperture microscope objective, using a digital micro-mirror device (DMD). We show that, after correcting the optical aberrations, light is confined within a spot of sub-micron lateral size and ∼100 nm axial depth above the coverslip, resulting in a volume of illumination drastically smaller than the one generated by a standard propagative focus. This evanescent focus is sufficient to induce a more intense and localized recruitment compared to a propagative focus on the optogenetic system CRY2-CIBN, improving the resolution of its pattern of activation.

Multimodal mapping of regional brain vulnerability to focal cortical dysplasia.

Focal cortical dysplasia (FCD) type II is a highly epileptogenic developmental malformation and a common cause of surgically treated drug-resistant epilepsy. While clinical observations suggest frequent occurrence in the frontal lobe, mechanisms for such propensity remain unexplored. Here, we hypothesized that cortex-wide spatial associations of FCD distribution with cortical cytoarchitecture, gene expression and organizational axes may offer complementary insights into processes that predispose given cortical regions to harbour FCD. We mapped the cortex-wide MRI distribution of FCDs in 337 patients collected from 13 sites worldwide. We then determined its associations with (i) cytoarchitectural features using histological atlases by Von Economo and Koskinas and BigBrain; (ii) whole-brain gene expression and spatiotemporal dynamics from prenatal to adulthood stages using the Allen Human Brain Atlas and PsychENCODE BrainSpan; and (iii) macroscale developmental axes of cortical organization. FCD lesions were preferentially located in the prefrontal and fronto-limbic cortices typified by low neuron density, large soma and thick grey matter. Transcriptomic associations with FCD distribution uncovered a prenatal component related to neuroglial proliferation and differentiation, likely accounting for the dysplastic makeup, and a postnatal component related to synaptogenesis and circuit organization, possibly contributing to circuit-level hyperexcitability. FCD distribution showed a strong association with the anterior region of the antero-posterior axis derived from heritability analysis of interregional structural covariance of cortical thickness, but not with structural and functional hierarchical axes. Reliability of all results was confirmed through resampling techniques. Multimodal associations with cytoarchitecture, gene expression and axes of cortical organization indicate that prenatal neurogenesis and postnatal synaptogenesis may be key points of developmental vulnerability of the frontal lobe to FCD. Concordant with a causal role of atypical neuroglial proliferation and growth, our results indicate that FCD-vulnerable cortices display properties indicative of earlier termination of neurogenesis and initiation of cell growth. They also suggest a potential contribution of aberrant postnatal synaptogenesis and circuit development to FCD epileptogenicity.

Imaging through a square multimode fiber by scanning focused spots with the memory effect.

The existence of a shift-shift memory effect in square waveguides, whereby any translation of the input field induces translations in the output field in four symmetrical directions, has been previously observed by correlation measurements. Here we demonstrate that this memory effect is also observed in real space and can be put to use for imaging purposes. First, a focus is created at the output of a square-core multimode fiber, by wavefront shaping based on feedback from a guide-star. Then, because of the memory effect, four symmetrical spots can be scanned at the fiber output by shifting the wavefront at the fiber input. We demonstrate that this property can be exploited to perform fluorescence imaging through the multimode fiber, without requiring the measurement of a transmission matrix.

Low-Temperature Gas-Phase Kinetics of Ethanol-Methanol Heterodimer Formation.

The structures of gas-phase noncovalently bound clusters have long been studied in supersonic expansions. This method of study, while providing a wealth of information about the nature of noncovalent bonds, precludes observation of the formation of the cluster, as the clusters form just after the orifice of the pulsed valve. Here, we directly observe formation of ethanol-methanol dimers via microwave spectroscopy in a controlled cryogenic environment. Time profiles of the concentration of reagents in the cell yielded gas-phase reaction rate constants of kMe-g = (2.8 ± 1.4) × 10-13 cm3 molecule-1 s-1 and kMe-t = (1.6 ± 0.8) × 10-13 cm3 molecule-1 s-1 for the pseudo-second-order ethanol-methanol dimerization reaction at 8 K. The relaxation cross section between the gauche and trans conformers of ethanol was also measured using the same technique. In addition, thermodynamic relaxation between conformers of ethanol over time allowed for selection of conformer stoichiometry in the ethanol-methanol dimerization reaction, but no change in the ratio of dimer conformers was observed with changing ethanol monomer stoichiometry.

Inter-individual variability in dorsal stream dynamics during word production.

The current standard model of language production involves a sensorimotor dorsal stream connecting areas in the temporo-parietal junction with those in the inferior frontal gyrus and lateral premotor cortex. These regions have been linked to various aspects of word production such as phonological processing or articulatory programming, primarily through neuropsychological and functional imaging group studies. Most if not all the theoretical descriptions of this model imply that the same network should be identifiable across individual speakers. We tested this hypothesis by quantifying the variability of activation observed across individuals within each dorsal stream anatomical region. This estimate was based on electrical activity recorded directly from the cerebral cortex with millisecond accuracy in awake epileptic patients clinically implanted with intracerebral depth electrodes for pre-surgical diagnosis. Each region's activity was quantified using two different metrics-intra-cerebral evoked related potentials and high gamma activity-at the level of the group, the individual and the recording contact. The two metrics show simultaneous activation of parietal and frontal regions during a picture naming task, in line with models that posit interactive processing during word retrieval. They also reveal different levels of between-patient variability across brain regions, except in core auditory and motor regions. The independence and non-uniformity of cortical activity estimated through the two metrics push the current model towards sub-second and sub-region explorations focused on individualized language speech production. Several hypotheses are considered for this within-region heterogeneity.

Combining Fluorescence Fluctuations and Photobleaching to Quantify Surface Density.

We have established a self-calibrated method, called pbFFS for photobleaching fluctuation fluorescence spectroscopy, which aims to characterize molecules or particles labeled with an unknown distribution of fluorophores. Using photobleaching as a control parameter, pbFFS provides information on the distribution of fluorescent labels and a reliable estimation of the absolute density or concentration of these molecules. We present a complete theoretical derivation of the pbFFS approach and experimentally apply it to measure the surface density of a monolayer of fluorescently tagged streptavidin molecules, which can be used as a base platform for biomimetic systems. The surface density measured by pbFFS is consistent with the results of spectroscopic ellipsometry, a standard surface technique. However, pbFFS has two main advantages: it enables in situ characterization (no dedicated substrates are required) and can be applied to low masses of adsorbed molecules, which we demonstrate here by quantifying the density of biotin-Atto molecules that bind to the streptavidin layer. In addition to molecules immobilized on a surface, we also applied pbFFS to molecules diffusing in solution, to confirm the distribution of fluorescent labels found on a surface. Hence, pbFFS provides a set of tools for investigating the molecules labeled with a variable number of fluorophores, with the aim of quantifying either the number of molecules or the distribution of fluorescent labels, the latter case being especially relevant for oligomerization studies.

Optical nanotopography of fluorescent surfaces by axial position modulation.

We present an optical method that combines confocal microscopy with position modulation to perform axial tracking and topographic imaging of fluorescent surfaces. Using a remote focusing system, the confocal observation volume is oscillated in the axial direction. The resulting modulation of the detected signal is used as a feedback to precisely control the distance to an object of interest. The accuracy of this method is theoretically analyzed and the axial-locking accuracy is experimentally evaluated. Topographic imaging is demonstrated on fluorescently coated beads and fixed cells. This microscope allows for nanometric topography or tracking of dynamic fluorescent surfaces.

Multilobar unilateral hypoplasia with emphasis on the posterior quadrant and severe epilepsy in children with FCD ILAE Type 1A.

OBJECTIVE: Focal cortical dysplasia (FCD) Type 1 and its three subtypes have yet not been fully characterized at the clinical, anatomopathological, and molecular level (International League Against Epilepsy [ILAE] FCD classification from 2011). We aimed to describe the clinical phenotype of patients with histopathologically confirmed FCD1A obtained from a single epilepsy center between 2002 and 2016. METHODS: Medical records were retrieved from the hospital's archive. Results from electroencephalography (EEG) video recordings, neuroimaging, and histopathology were reevaluated. Magnetic resonance imaging (MRI) post-processing was retrospectively performed in nine patients. DNA methylation studies were carried out from archival surgical brain tissue in 11 patients. RESULTS: Nineteen children with a histopathological diagnosis of FCD1A were included. The average onset of epilepsy was 0.9 years (range 0.2-10 years). All children had severe cognitive impairment and one third had mild motor deficits, yet fine finger movements were preserved in all patients. All patients had daily seizures, being drug resistant from disease onset. Interictal electroencephalography revealed bilateral multi-regional epileptiform discharges. Interictal status epilepticus was observed in 8 and countless subclinical seizures in 11 patients. Regional continuous irregular slow waves were of higher lateralizing and localizing yield than spikes. Posterior background rhythms were normal in 16 of 19 children. Neuroimaging showed unilateral multilobar hypoplasia and increased T2-FLAIR signals of the white matter in 18 of 19 patients. All children underwent tailored multilobar resections, with seizure freedom achieved in 47% (Engel class I). There was no case with frontal involvement without involvement of the posterior quadrant by MRI and histopathology. DNA methylation profiling distinguished FCD1A samples from all other epilepsy specimens and controls. SIGNIFICANCE: We identified a cohort of young children with drug resistance from seizure onset, bad EEG with posterior emphasis, lack of any focal neurological deficits but severe cognitive impairment, subtle hypoplasia of the epileptogenic area on MRI, and histopathologically defined and molecularly confirmed by DNA methylation analysis as FCD ILAE Type 1A.