FAN1 controls mismatch repair complex assembly via MLH1 retention to stabilize CAG repeat expansion in Huntington's disease

CAG repeat expansion in the HTT gene drives Huntington’s disease (HD) pathogenesis and is modulated by DNA damage repair pathways. In this context, the interaction between FAN1, a DNA-structure-specific nuclease, and MLH1, member of the DNA mismatch repair pathway (MMR), is not defined. Here, we identify a highly conserved SPYF motif at the N terminus of FAN1 that binds to MLH1. Our data support a model where FAN1 has two distinct functions to stabilize CAG repeats. On one hand, it binds MLH1 to restrict its recruitment by MSH3, thus inhibiting the assembly of a functional MMR complex that would otherwise promote CAG repeat expansion. On the other hand, it promotes accurate repair via its nuclease activity. These data highlight a potential avenue for HD therapeutics in attenuating somatic expansion

Mass spectrometry-based absolute quantification of 20S proteasome status for controlled ex-vivo expansion of Human Adipose-derived Mesenchymal Stromal/Stem Cells

The proteasome controls a multitude of cellular processes through protein degradation and has been identified as a therapeutic target in oncology. However, our understanding of its function and the development of specific modulators are hampered by the lack of a straightforward method to determine the overall proteasome status in biological samples. Here, we present a method to determine the absolute quantity and stoichiometry of ubiquitous and tissue-specific human 20S proteasome subtypes based on a robust, absolute SILAC-based multiplexed LC-Selected Reaction Monitoring (SRM) quantitative mass spectrometry assay with high precision, accuracy, and sensitivity. The method was initially optimized and validated by comparison with a reference ELISA assay and by analyzing the dynamics of catalytic subunits in HeLa cells following IFNγ-treatment and in range of human tissues. It was then successfully applied to reveal IFNγ- and O2-dependent variations of proteasome status during primary culture of Adipose-derived-mesenchymal Stromal/Stem Cells (ADSCs). The results show the critical importance of controlling the culture conditions during cell expansion for future therapeutic use in humans. We hypothesize that a shift from the standard proteasome to the immunoproteasome could serve as a predictor of immunosuppressive and differentiation capacities of ADSCs and, consequently, that quality control should include proteasomal quantification in addition to examining other essential cell parameters. The method presented also provides a new powerful tool to conduct more individualized protocols in cancer or inflammatory diseases where selective inhibition of the immunoproteasome has been shown to reduce side effects

Multispectral total-variation reconstruction applied to lens-free microscopy

International audienceLens-free microscopy multispectral acquisitions are processed with an inverse problem approach: a multispectral total variation criterion is defined and minimized with the conjugate gradients method. Reconstruction results show that the method is efficient to recover the phase image of densely packed cells

Dynamics of cell and tissue growth acquired by means of extended field of view lensfree microscopy

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Vliv klomipraminu a risperidonu na učení a flexibilitu u animálního modelu obsedantně kompulzivní poruchy

Chronic sensitization of dopamine D2/D3 receptors by agonist quinpirole (QNP) induces compulsive checking behaviour in rats, which is considered an animal model of obsessive-compulsive disorder (OCD). Previous study revealed deficit in cognitive flexibility in QNP sensitized rats. This thesis focused on determining if this cognitive flexibility deficit is ameliorated by co-administration of clomipramine (CMI), risperidone (RIS) or combination of both (CMI+RIS) to QNP treatment. Aversively motivated active place avoidance task on a Carousel maze with reversal was used. The number of entrances into a to-be-avoided shock sector was evaluated as measure of performance. Six treatment groups were used: control group, QNP group, CMI group, QNP/CMI combination, QNP/RIS combination and QNP/CMI/RIS combination. Surprisingly, when compared alone, significantly worse acquisition was observed for QNP group compared to control group. However, similarly to previous study, QNP group had a worse performance in a first reversal session compared to control group. When all groups were compared, only QNP/CMI group had worse initial learning compared to control group. In reversal learning, only QNP treated group had a significantly more entrances than control group in first reversal session. Results suggest that co-treatment..

Visualization 4: Dynamics of cell and tissue growth acquired by means of extended field of view lensfree microscopy

Real-time PIV lensfree sequence Originally published in Biomedical Optics Express on 01 February 2016 (boe-7-2-512

Visualization 5: Dynamics of cell and tissue growth acquired by means of extended field of view lensfree microscopy

Real-time PIV lensfree sequence Originally published in Biomedical Optics Express on 01 February 2016 (boe-7-2-512

Lensfree video microscopy: high throughput monitoring and cell tracking of 2D cell cultures

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Computational Strategies and Challenges for Using Native Ion Mobility Mass Spectrometry in Biophysics and Structural Biology

Native mass spectrometry (MS) allows the interrogation of structural aspects of macromolecules in the gas phase, under the premise of having initially maintained their solution-phase noncovalent interactions intact. In the more than 25 years since the first reports, the utility of native MS has become well established in the structural biology community. The experimental and technological advances during this time have been rapid, resulting in dramatic increases in sensitivity, mass range, resolution, and complexity of possible experiments. As experimental methods have improved, there have been accompanying developments in computational approaches for analyzing and exploiting the profusion of MS data in a structural and biophysical context. In this perspective, we consider the computational strategies currently being employed by the community, aspects of best practice, and the challenges that remain to be addressed. Our perspective is based on discussions within the European Cooperation in Science and Technology Action on Native Mass Spectrometry and Related Methods for Structural Biology (EU COST Action BM1403), which involved participants from across Europe and North America. It is intended not as an in-depth review but instead to provide an accessible introduction to and overview of the topic—to inform newcomers to the field and stimulate discussions in the community about addressing existing challenges. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05792) focuses on software tools available to help researchers tackle some of the challenges enumerated here