Microcollimator for Micrometer-Wide Stripe Irradiation of Cells Using 20–30 keV X Rays

The exposure of subnuclear compartments of cells to ionizing radiation is currently not trivial. We describe here a collimator for micrometer-wide stripe irradiation designed to work with conventional high-voltage X-ray tubes and cells cultured on standard glass cover slips. The microcollimator was fabricated by high-precision silicon micromachining and consists of X-ray absorbing chips with grooves of highly controlled depths, between 0.5-10 mum, along their surfaces. These grooves form X-ray collimating slits when the chips are stacked against each other. The use of this device for radiation biology was examined by irradiating human cells with X rays having energies between 20-30 keV. After irradiation, p53 binding protein 1 (53BP1), a nuclear protein that is recruited at sites of DNA double-strand breaks, clustered in lines corresponding to the irradiated stripes

Lupus induit et DRESS : effet indésirable rare mais redoutable de la mésalazine

La survenue d’un syndrome inflammatoire d’étiologie indéterminée chez un patient traité par mésalazine, m’a conduit à réfléchir sur les effets indésirables immunomédiés de ce traitement. La mésalazine, souvent utilisée comme traitement de première intention de la colite ulcéreuse, est associée à des effets indésirables systémiques dont ont fait état plusieurs publications. La plupart ont évoqué un syndrome d'hypersensibilité médicamenteuse avec comme manifestation principale un état fébrile et des symptômes gastro-intestinaux, mais plus rarement un syndrome d'hypersensibilité médicamenteuse avec éosinophilie et symptômes systémiques aussi nommé DRESS (drug reaction with eosinophilia and systemic symptoms). Quelques auteurs ont décrit des syndromes lupiques avec des caractéristiques clinico-biologiques limitées. Le tableau clinico-biologique décrit chez ce patient, que j’ai suivi en hospitalisation, associant une pleuropéricardite, une néphrite et des polyarthromyalgies induit par la mésalazine s'est résolu à l'arrêt du traitement sans recours aux stéroïdes. En partant de ce cas clinique, j’ai effectué une analyse de la littérature, venant étayer notre hypothèse diagnostique principale, d’un syndrome d'hypersensibilité médicamenteuse avec manifestations lupiques induit par la mésalazine.</p

Mechanism of recruitment of p53-binding protein 1 (53BP1) to DNA double-strand breaks

p53-binding protein 1 (53BP1) is a conserved nuclear protein that is rapidly recruited to sites of DNA double-strand breaks (DSBs) following DNA damage. Here, we show that the mechanism of recruitment involves recognition of a methylated lysine in histone H3, namely K79, which becomes exposed following break-induced chromatin relaxation. We have solved the crystal structure of the domain necessary for its recruitment, and found that it consists of two tandem Tudor domains with a deep pocket at their interface formed by evolutionarily conserved hydrophobic residues. Single amino acid substitutions of residues that form the walls of the 53BP1 hydrophobic pocket abrogate both binding to methylated histone H3 and also recruitment to sites of DNA DSBs. Second, depletion of Dot1L, the enzyme that methylates histone H3 on Lys79 in vivo, inhibits recruitment of 53BP1 to sites of DNA DSBs. Methylation of histone H3 Lys79 is not enhanced in response to DNA damage, supporting the model that a DNA DSB could lead to disruption of higher order chromatin structure, resulting in nucleosome unstacking and exposure of methylated Lys79 of histone H3. We later demonstrate that the tandem Tudor domain alone is not sufficient for recruitment, and that an upstream oligomerization domain is required for this function. We have mapped the boundaries of this new domain and find it to comprise approximately 40 residues, lying at a distance of around 200 residues upstream of the Tudor. Deletion and point mutations in this domain abolish recruitment to DSBs, but this is rescued by introducing an exogenous oligomerization domain upstream of the Tudor. Further, deletion mutants that are foci-proficient migrate as higher-order oligomers on a gel filtration column, while those that are foci-deficient migrate as monomers. We propose that 53BP1 oligomerization facilitates its interaction with relaxed chromatin that results from DNA breaks

An Oligomerized 53BP1 Tudor Domain Suffices for Recognition of DNA Double-Strand Breaks▿

53BP1, the vertebrate ortholog of the budding yeast Rad9 and fission yeast Crb2/Rhp9 checkpoint proteins, is recruited rapidly to sites of DNA double-strand breaks (DSBs). A tandem tudor domain in human 53BP1 that recognizes methylated residues in the histone core is necessary, but not sufficient, for efficient recruitment. By analysis of deletion mutants, we identify here additional elements in 53BP1 that facilitate recognition of DNA DSBs. The first element corresponds to an independently folding oligomerization domain. Replacement of this domain with heterologous tetramerization domains preserves the ability of 53BP1 to recognize DNA DSBs. A second element is only about 15 amino acids long and appears to be a C-terminal extension of the tudor domain, rather than an independently functioning domain. Recruitment of 53BP1 to sites of DNA DSBs is facilitated by histone H2AX phosphorylation and ubiquitination. However, none of the 53BP1 domains/elements important for recruitment are known to bind phosphopeptides or ubiquitin, suggesting that histone phosphorylation and ubiquitination regulate 53BP1 recruitment to sites of DNA DSBs indirectly

Odyssey of a Misclassified Genomic Variant: Insight from an Incidental Finding Assessment

Genetic evaluation of a teenager with seizure found no pathogenic variant in a large gene panel, but an incidental likely pathogenic HNF4A variant, deemed to cause MODY1 diabetes. Diabetes history was absent and glycated hemoglobin normal, but serum calcium was severely low, with abnormally high parathyroid hormone. Thus, pseudohypoparathyroidism was suspected and confirmed by molecular genetic testing. Calcium and calcitriol supplementation led to calcium normalization and neurological symptom improvement. Given the absence of personal or family diabetes history, the HNF4A variant was reassessed and found to encode an alternative transcript with poor expression and activity levels, hence downgraded on expert advice from ‘likely pathogenic’ to ‘likely benign’. Besides illustrating the importance of structured medical workup before launching extensive targeted exome sequencing, this case highlights the need for caution in incidental finding interpretation in patients lacking compatible phenotype or family history, and the value of expert advice in such variant interpretation

A Model to Investigate Single-Strand DNA Responses in G1 Human Cells via a Telomere-Targeted, Nuclease-Deficient CRISPR-Cas9 System

<div><p>DNA replication stress has the potential to compromise genomic stability and, therefore, cells have developed elaborate mechanisms to detect and resolve problems that may arise during DNA replication. The presence of single-stranded DNA (ssDNA) is often associated with DNA replication stress and serves as a signal for both checkpoint and repair responses. In this study, we exploited a CRISPR-Cas9 system to induce regions of ssDNA in the genome. Specifically, single-guide RNAs bearing sequence complementarity to human telomeric repeats, were used to target nuclease-deficient Cas9 (dCas9) to telomeres. Such targeting was associated with the formation of DNA-RNA hybrids, leaving one telomeric DNA strand single-stranded. This ssDNA then recruited DNA repair and checkpoint proteins, such as RPA, ATRIP, BLM and Rad51, at the telomeres. Interestingly, targeting of all these proteins to telomeric ssDNA was observed even in cells that were in the G1 phase of the cell cycle. Therefore, this system has the potential to serve as a platform for further investigation of DNA replication stress responses at specific loci in the human genome and in all phases of the cell cycle.</p></div

Specific recruitment to telomeres of sgTelomere RNA, colocalizing with TRF1.

<p>(a) Schematic representation of the various constructs used in this study: 3XFLAG-tagged dCas9 and a separately translated GFP marker, containing sgRNA lacking a cognate sequence in the human genome (sgControl), sgRNA corresponding to the mucin gene loci (sgMUC4-E3), or telomere sgRNA (sgTelomere). (b) An untransfected U2OS cell (upper panel) showing no detectable FLAG signal by immunofluorescence; sgTelomere transfected cell (second panel) showing nuclear FLAG foci colocalizing with TRF1; sgControl transfected cell (third panel) showing diffuse cellular FLAG signal; sgMUC4-E3 transfected cell (bottom panel) with no observable mucin gene loci.</p

Drug-induced hypersensitivity syndrome with lupus manifestations due to mesalazine in a patient with ulcerative colitis

Mesalazine is often used as first-line therapy for ulcerative colitis. Several reports have pointed to systemic adverse reactions associated with this drug. Most have evoked a drug-induced hypersensitivity syndrome, while some have described lupus syndromes but with limited clinical and varied biological features. A 75-year-old man presented with fever, dyspnoea, chest pain, polyarthralgia, and myalgia, following mesalazine introduction. Clinical symptoms and low-titre positive antihistone antibodies disappeared after mesalazine withdrawal without recourse to steroids. Pericardial effusion and 8F-fluorodeoxyglucose uptake on positron emission tomography/CT scan, and glomerular haematuria and proteinuria also disappeared. Cytokine-lymphocyte transformation tests showed a strong sensitisation pattern with interleukin-5 production. This case advances our knowledge of the mechanism of mesalazine-induced adverse effects, namely via drug-induced hypersensitivity with lupus manifestations, which we are the first to report

Induction of ssDNA at human telomeres.

<p>(a) Schematic description of the experimental setup, with or without 8h treatment of 10 μM ATR inhibitor. (b) Specific colocalization of sgTelomere FLAG-dCas9 with RPA70 (upper panel), RPA32 pS33 (middle panel) and ATRIP (bottom panel). (c) Quantification of cells according to foci colocalization observed in (b). RPA70, ATRIP, and RPA32 phosphorylated at S33 colocalized with sgTelomere FLAG-dCas9 in approximately 72%, 55%, and 63% of all counted cells. A total number of 120 cells per condition were counted. (d) Quantification of cells according to foci colocalization with or without treatment with 10 μM ATR inhibitor (ATRi). There was no significant effect on RPA recruitment to ssDNA (86% versus 72% without ATRi, p = 0.10), or on ATRIP recruitment to ssDNA (58% versus 55% without ATRi, p = 0.83). However, there is a significant decrease (from 63% to 38%) in the number of RPA32-pS33 foci colocalizing with FLAG-dCas9 (p<0.008). A total number of 35 cells per condition were counted. (e) Quantification of TRF1-foci-positive cells showing RPA70 colocalization. Untransfected, sgControl-, and sgTelomere-transfected cells showed, respectively, 5%, 4%, and 50% colocalization. A total number of 100 cells were counted (p<0.00001).</p

Involvement of DNA repair pathways.

<p>(a) Specific colocalization of sgTelomere FLAG-dCas9 with Bloom helicase (upper panel), Rad51 (second panel), gamma-H2AX (third panel), and RecQL4 (bottom panel). (b) Quantification of cells according to foci colocalization observed in (a). In nearly all cells, FLAG-dCas9 colocalized with Bloom helicase (95%) and Rad51 (93%). No colocalization was observed with gamma-H2AX or RecQL4. A total number of 100 cells were counted (p<0.00001).</p