Bis-locked nucleic acids : a new tool for double helix invasion

Deoxyribonucleic acid (DNA) is a macromolecule that contains the information for all living organisms to achieve important cellular processes such as growth and replication. DNA consists of two strands coiled around each other to form a double helix. Several interactions including Watson-Crick pairing and stacking between the bases stabilize the double helix. To ensure the integrity of genetic information, the bases are on the inside the double helix whereas the phosphate and sugar groups are on the outside. Thus, most DNA information is not accessible until DNA is unwound prior to replication or transcription. In cells, enzymes called helicases achieve DNA strands separation. The anti-gene strategy aims to introduce a short single-stranded oligonucleotide (ON) to bind genomic DNA at a specific site to block the mRNA transcription. As a result, mRNA and protein expression for a specific gene are expected to be reduced, which could be beneficial in certain clinical contexts. However, this therapeutic strategy is hampered by the low stability of the DNA binding, the difficulty to reach the genomic target and to block the RNA polymerase, suggesting the needs for new DNA binding ONs. In this thesis, we developed clamp-ONs, bis-locked nucleic (bisLNAs), binding via Hoogsteen and Watson-Crick interactions. In paper I, we assessed their binding characteristics and demonstrated their ability to invade supercoiled DNA at intranuclear pH and salt conditions. In paper II, we investigated the step-by-step mechanism for bisLNA invasion. Based on this mechanism, we designed a new generation of bisLNAs using nonnatural modifications such as a stacking linker and 2´-glycylamino LNAs. In paper III, we investigated how the bisLNAs invade in a more complex environment using rolling circle amplification for detection

Correlations between soluble alpha/beta forms of amyloid precursor protein and Abeta38, 40 and 42 in human cerebrospinal fluid

International audienceCerebrospinal fluid (CSF) biomarkers are now widely used for diagnosis of Alzheimer disease (AD) in atypical clinical forms, for differential and early diagnosis, or for stratification of patients in clinical trials. Among these biomarkers, different forms of amyloid peptides (Aβ) produced by the cleavage of a transmembrane precursor protein called APP (amyloid precursor protein) have a major role. Aβ peptides exist in different length the most common ones having 40 (Aβ40), 42 (Aβ42), or 38 (Aβ38) amino acids in length. APP processing by gamma-secretase releases also an amino-terminal secreted fragment called sAβPP-beta while an alternative nonamyloidogenic cleavage of APP, through an alpha-secretase, liberates another fragment called sAβPP-alpha. To decipher the molecular and pathological mechanisms leading to the production and the detection of these entities is essential for the comprehension and the prevention of AD. In this report, we present the results of the Keywords: Biomarkers CSF Soluble amyloid precursor proteins Aβ fragment peptides Alzheimer disease Dementi

Decreased sAβPPβ, Aβ38, and Aβ40 Cerebrospinal Fluid Levels in Frontotemporal Dementia.

International audienceTo improve the etiological diagnosis of neurodegenerative dementias like Alzheimer's disease (AD) or frontotemporal dementia (FTD), we evaluated the value of individual and combined measurements of the following relevant cerebrospinal fluid (CSF) biomarkers: Tau, 181p-Tau, Aβ38, Aβ40, Aβ42, sAβPPα, and sAβPPβ. This study conducted in two centers included patients with FTD (n = 34), AD (n = 52), as well as a control group of persons without dementia (CTRL, n = 42). Identical clinical criteria and pre-analytical conditions were used while CSF biomarkers were measured using commercial single and multiplex quantitative immunoassays. Thorough statistical analyses, including ROC curves, logistic regressions, and decision trees, were performed. We validated in AD the specific increase of p-Tau levels and the decrease of Aβ42 levels, two biological hallmarks of this disease. Tau concentrations were highest in AD and intermediate in FTD when compared to CTRL. The most interesting results were obtained by focusing on amyloid biomarkers as we found out in FTD a significant decrease of sAβPPβ, Aβ38, and Aβ40 levels. Aβ38 in particular was the most useful biomarker to differentiate FTD subjects from the CTRL population. Combining p-Tau and Aβ38 led us to correctly classifying FTD patients with sensitivity at 85% and specificity at 82%. Significant changes in amyloid biomarkers, particularly for Aβ38, are therefore seen in FTD. This could be quite useful for diagnosis purposes and it might provide additional evidence on the interrelationship between Tau and AβPP biology which understanding is essential to progress towards optimal therapeutic and diagnostic approaches of dementia

Gene Tagging with the CRISPR-Cas9 System to Facilitate Macromolecular Complex Purification

The need to generate modified cell lines that express tagged proteins of interest has become increasingly important. Here, we describe a detailed protocol for facile CRISPR/Cas9-mediated gene tagging and isolation of modified cells. In this protocol, we combine two previously published strategies that promote CRISPR/Cas9-mediated gene tagging: using chemically modified single-stranded oligonucleotides as donor templates and a co-selection strategy targeting the ATP1A1 gene at the same time as the gene of interest. Altogether, the protocol proposed here is both easier and saves time compared to other approaches for generating cells that express tagged proteins of interest, which is crucial to purify native complex from human cells

Parkinsonian Patients Requiring Proteasome Inhibitors for Multiple Myeloma: Exceptional Circumstances Call for Extra Caution

Feedback on the management of patients with both Parkinson’s disease (PD) and multiple myeloma (MM) is not common in the literature, and we would like to report an original possible adverse event (AE) of MM chemotherapy: an increased PD severity induced by ixazomib. The patient, a 68-year-old man with PD (Hoehn and Yahr (HY) stage 3) was effectively and stably treated for 14 years with dopaminergic therapy (levodopa equiva25 lent daily dose (LEDD) 1275 mg). His symptomatic MM was first treated by lenalidomide (25 mg) with dexamethasone (Rd protocol) for 19 months. Then, a second-line therapy combined the proteasome inhibitor (PI) ixazomib (4 mg) with lenalidomide (20 mg) and dexamethasone (IRd protocol, 4-week cycle) due to MM progression

Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System and Double-Stranded DNA Donors

International audienceMacromolecular complexes govern the majority of biological processes and are of great biomedical relevance as factors that perturb interaction networks underlie a number of diseases, and inhibition of protein-protein interactions is a common strategy in drug discovery. Genome editing technologies enable precise modifications in protein coding genes in mammalian cells, offering the possibility to introduce affinity tags or fluorescent reporters for proteomic or imaging applications in the bona fide cellular context. Here we describe a streamlined procedure which uses the CRISPR/Cas9 system and a double-stranded donor plasmid for efficient generation of homozygous endogenously GFP-tagged human cell lines. Establishing cellular models that preserve native genomic regulation of the target protein is instrumental to investigate protein localization and dynamics using fluorescence imaging but also to affinity purify associated protein complexes using anti-GFP antibodies or nanobodies

Automatic Liver Viability Scoring with Deep Learning and Hyperspectral Imaging.

Hyperspectral imaging (HSI) is a non-invasive imaging modality already applied to evaluate hepatic oxygenation and to discriminate different models of hepatic ischemia. Nevertheless, the ability of HSI to detect and predict the reperfusion damage intraoperatively was not yet assessed. Hypoxia caused by hepatic artery occlusion (HAO) in the liver brings about dreadful vascular complications known as ischemia-reperfusion injury (IRI). Here, we show the evaluation of liver viability in an HAO model with an artificial intelligence-based analysis of HSI. We have combined the potential of HSI to extract quantitative optical tissue properties with a deep learning-based model using convolutional neural networks. The artificial intelligence (AI) score of liver viability showed a significant correlation with capillary lactate from the liver surface (r = -0.78, p = 0.0320) and Suzuki's score (r = -0.96, p = 0.0012). CD31 immunostaining confirmed the microvascular damage accordingly with the AI score. Our results ultimately show the potential of an HSI-AI-based analysis to predict liver viability, thereby prompting for intraoperative tool development to explore its application in a clinical setting

Exercise does not activate the β3 adrenergic receptor–eNOS pathway, but reduces inducible NOS expression to protect the heart of obese diabetic mice

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Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression

Chromatin structure plays a significant role in the regulation of gene expression. Here the authors show that TFIIH interacts with the histone acetyl transferase KAT2A and recruits the ATAC/hSAGA complexes to chromatin; and that loss of xeroderma pigmentosum group B (XPB) function results in chromatin decondensation and increased gene expression through activation of KAT2A

Quality assessment in light microscopy for routine use through simple tools and robust metrics

International audienceAlthough there is a need to demonstrate reproducibility in light microscopy acquisitions, the lack of standardized guidelines monitoring microscope health status over time has so far impaired the widespread use of quality control (QC) measurements. As scientists from 10 imaging core facilities who encounter various types of projects, we provide affordable hardware and open source software tools, rigorous protocols, and define reference values to assess QC metrics for the most common fluorescence light microscopy modalities. Seven protocols specify metrics on the microscope resolution, field illumination flatness, chromatic aberrations, illumination power stability, stage drift, positioning repeatability, and spatial-temporal noise of camera sensors. We designed the MetroloJ_QC ImageJ/Fiji Java plugin to incorporate the metrics and automate analysis. Measurements allow us to propose an extensive characterization of the QC procedures that can be used by any seasoned microscope user, from research biologists with a specialized interest in fluorescence light microscopy through to core facility staff, to ensure reproducible and quantifiable microscopy results