Histone H3.3G34-Mutant Interneuron Progenitors Co-opt PDGFRA for Gliomagenesis.

Histone H3.3 glycine 34 to arginine/valine (G34R/V) mutations drive deadly gliomas and show exquisite regional and temporal specificity, suggesting a developmental context permissive to their effects. Here we show that 50% of G34R/V tumors (n = 95) bear activating PDGFRA mutations that display strong selection pressure at recurrence. Although considered gliomas, G34R/V tumors actually arise in GSX2/DLX-expressing interneuron progenitors, where G34R/V mutations impair neuronal differentiation. The lineage of origin may facilitate PDGFRA co-option through a chromatin loop connecting PDGFRA to GSX2 regulatory elements, promoting PDGFRA overexpression and mutation. At the single-cell level, G34R/V tumors harbor dual neuronal/astroglial identity and lack oligodendroglial programs, actively repressed by GSX2/DLX-mediated cell fate specification. G34R/V may become dispensable for tumor maintenance, whereas mutant-PDGFRA is potently oncogenic. Collectively, our results open novel research avenues in deadly tumors. G34R/V gliomas are neuronal malignancies where interneuron progenitors are stalled in differentiation by G34R/V mutations and malignant gliogenesis is promoted by co-option of a potentially targetable pathway, PDGFRA signaling.

Mechanisms and therapeutic implications of hypermutation in gliomas.

A high tumour mutational burden (hypermutation) is observed in some gliomas1-5; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer.

Aberrant DNA repair reveals a vulnerability in histone H3.3-mutant brain tumors.

Pediatric high-grade gliomas (pHGG) are devastating and incurable brain tumors with recurrent mutations in histone H3.3. These mutations promote oncogenesis by dysregulating gene expression through alterations of histone modifications. We identify aberrant DNA repair as an independent mechanism, which fosters genome instability in H3.3 mutant pHGG, and opens new therapeutic options. The two most frequent H3.3 mutations in pHGG, K27M and G34R, drive aberrant repair of replication-associated damage by non-homologous end joining (NHEJ). Aberrant NHEJ is mediated by the DNA repair enzyme polynucleotide kinase 3'-phosphatase (PNKP), which shows increased association with mutant H3.3 at damaged replication forks. PNKP sustains the proliferation of cells bearing H3.3 mutations, thus conferring a molecular vulnerability, specific to mutant cells, with potential for therapeutic targeting.

Fast numerical autofocus of multispectral complex fields in digital holographic microscopy with a criterion based on the phase in the Fourier domain.

The knowledge of the complex amplitude of optical fields, that is, both quantitative phase and intensity, enables numeric reconstruction along the optical axis. Nonetheless, a criterion is required for autofocusing. This Letter presents a robust and rapid refocusing criterion suitable for color interferometric digital holographic microscopy, and, more generally, for applications where complex amplitude is known for at least two different wavelengths. This criterion uses the phase in the Fourier domain, which is compared among wavelengths. It is applicable whatever the nature of the observed object: opaque, refractive, or both mixed. The method is validated with simulated and experimental holograms.

Digital holographic microscopy as a tool to study the thermal shape fluctuations of lipid vesicles.

The bending elasticity modulus of lipid membranes is obtained by applying for the first time, to the best of our knowledge, a novel experimental technique based on digital holographic microscopy. The fluctuations of the radius with time were extracted by tracking and measuring the optical thickness at the vesicle poles. The temporal autocorrelation function of the vesicle diameter computed for each of the studied vesicles was then fitted with the theoretical expression to deduce the membrane's tension and bending constant. For the bending elasticity modulus of SOPC bilayers, the value of (0.93 ± 0.03) × 10(-12) erg was obtained. This result is in accordance with values previously obtained by means of other conventional methods for the same type of lipid membrane in the presence of sugar molecules in aqueous medium. The obtained results encourage the future development of the digital holographic microscopy as a technique suitable for the measurement of the bending elasticity of lipid membranes.

Quantitative assessment of noise reduction with partial spatial coherence illumination in digital holographic microscopy.

Improving image quality in digital holographic microscopy is achievable by using partial spatial coherence (PSC) illumination instead of fully coherent illumination. This Letter presents simple theoretical models to quantitatively assess the reduction of noise as a function of both the spatial coherence of the illumination and the defocus distance of the noise source. The first developed model states that the effect of the PSC can be studied by discretizing the field of view in the plane of the noise source. The second model, following a continuous approach, corroborates the discrete model and extends it. Experimental results confirm theoretical expectations.

Complex Drop Impact Morphology.

The aim of this work is to understand the changes in the observed phenomena during particle-laden drop impact. The impact of millimeter-size drops was investigated onto hydrophilic (glass) and hydrophobic (polycarbonate) substrates. The drops were dispersions of water and spherical and nearly iso-dense hydrophobic particles with diameters of 200 and 500 µm. The impact was studied by side and bottom view images in the range 150 ≤ We ≤ 750 and 7100 ≤ Re ≤ 16400. The particles suppressed the appearance of singular jetting and drop partial rebound but promoted splashing, receding breakup, and rupture. The drops with 200 µm particles spread in two phases: fast and slow, caused by inertial and capillary forces, respectively. Also, the increase in volume fraction of 200 µm particle led to a linear decrease in the maximum spreading factor caused by the inertia force on both hydrophilic and hydrophobic substrates. The explanation of this reduction was argued to be the result of energy dissipation through frictional losses between particles and the substrate.

High throughput holographic imaging-in-flow for the analysis of a wide plankton size range.

We developed a Digital Holographic Microscope (DHM) working with a partial coherent source specifically adapted to perform high throughput recording of holograms of plankton organisms in-flow, in a size range of 3 µm-300 µm, which is of importance for this kind of applications. This wide size range is achieved with the same flow cell and with the same microscope magnification. The DHM configuration combines a high magnification with a large field of view and provides high-resolution intensity and quantitative phase images refocusing on high sample flow rate. Specific algorithms were developed to detect and extract automatically the particles and organisms present in the samples in order to build holograms of each one that are used for holographic refocusing and quantitative phase contrast imaging. Experimental results are shown and discussed.

Refocusing based on amplitude analysis in color digital holographic microscopy.

A refocusing criterion adapted to red-green-blue (RGB) digital holographic microscopy is established. It is applicable for both amplitude and phase objects. This color criterion is based on a monochromatic criterion, using the integrated modulus amplitude. Simulated RGB holograms show the value of having color information, even for colorless samples; in addition, the position of the focus plane along the optical axis is determined more accurately. Simulations take into account both the numerical apertures of lenses and noise during the holographic process. We also implement an algorithm exponentially reducing the computation time required for detecting the focus plane. The method is validated on experimental holograms.

Color imaging-in-flow by digital holographic microscopy with permanent defect and aberration corrections.

Color imaging-in-flow of particles is performed using red-green-blue (RGB) digital holographic microscopy (DHM), whose sources are partially coherent. RGB DHM provides intensity and quantitative phase images in the three color channels, which is valuable for observing small objects in numerous fields. In-flow investigation on a large depth of field is made possible by the refocusing capability of DHM and has many potential applications. A method is also developed to automatically correct the color balance and compensate both intensity and phase defects and aberrations, providing high-quality imaging. Experimental results show color in-flow analysis of microplankton and confirm the efficiency of the correction method.