Tracking down protein-protein interactions via a FRET-system using site-specific thiol-labeling.

Förster resonance energy transfer is among the most popular tools to follow protein-protein interactions. Although limited to certain cases, site-specific fluorescent labeling of proteins via natural functions by means of chemical manipulations can redeem laborious protein engineering techniques. Herein we report on the synthesis of a heterobifunctional tag and its use in site-specific protein labeling studies aiming at exploring protein-protein interactions. The oxadiazole-methylsulfonyl functionality serves as a thiol specific warhead that enables easy and selective installation of fluorescent labels through a bioorthogonal motif. Mitogen activated protein kinase (MAPK14) and its substrate mitogen activated protein kinase activated kinase (MAPKAP2) or its docking motif, a 22 amino acid-long peptide fragment, were labeled with a donor and an acceptor, respectively. Evolution of strong FRET signals upon protein-protein interactions supported the specific communication between the partners. Using an efficient FRET pair allowed the estimation of dissociation constants for protein-protein and peptide-protein interactions (145 nM and 240 nM, respectively).

Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions.

This corrects the article DOI: 10.1038/onc.2016.243.

Fast and accurate mutation detection in whole genome sequences of multiple isogenic samples with IsoMut.

BACKGROUND: Detection of somatic mutations is one of the main goals of next generation DNA sequencing. A wide range of experimental systems are available for the study of spontaneous or environmentally induced mutagenic processes. However, most of the routinely used mutation calling algorithms are not optimised for the simultaneous analysis of multiple samples, or for non-human experimental model systems with no reliable databases of common genetic variations. Most standard tools either require numerous in-house post filtering steps with scarce documentation or take an unpractically long time to run. To overcome these problems, we designed the streamlined IsoMut tool which can be readily adapted to experimental scenarios where the goal is the identification of experimentally induced mutations in multiple isogenic samples. METHODS: Using 30 isogenic samples, reliable cohorts of validated mutations were created for testing purposes. Optimal values of the filtering parameters of IsoMut were determined in a thorough and strict optimization procedure based on these test sets. RESULTS: We show that IsoMut, when tuned correctly, decreases the false positive rate compared to conventional tools in a 30 sample experimental setup; and detects not only single nucleotide variations, but short insertions and deletions as well. IsoMut can also be run more than a hundred times faster than the most precise state of art tool, due its straightforward and easily understandable filtering algorithm. CONCLUSIONS: IsoMut has already been successfully applied in multiple recent studies to find unique, treatment induced mutations in sets of isogenic samples with very low false positive rates. These types of studies provide an important contribution to determining the mutagenic effect of environmental agents or genetic defects, and IsoMut turned out to be an invaluable tool in the analysis of such data.

Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions.

Loss-of-function mutations in the BRCA1 and BRCA2 genes increase the risk of cancer. Owing to their function in homologous recombination repair, much research has focused on the unstable genomic phenotype of BRCA1/2 mutant cells manifest mainly as large-scale rearrangements. We used whole-genome sequencing of multiple isogenic chicken DT40 cell clones to precisely determine the consequences of BRCA1/2 loss on all types of genomic mutagenesis. Spontaneous base substitution mutation rates increased sevenfold upon the disruption of either BRCA1 or BRCA2, and the arising mutation spectra showed strong and specific correlation with a mutation signature associated with BRCA1/2 mutant tumours. To model endogenous alkylating damage, we determined the mutation spectrum caused by methyl methanesulfonate (MMS), and showed that MMS also induces more base substitution mutations in BRCA1/2-deficient cells. Spontaneously arising and MMS-induced insertion/deletion mutations and large rearrangements were also more common in BRCA1/2 mutant cells compared with the wild-type control. A difference in the short deletion phenotypes of BRCA1 and BRCA2 suggested distinct roles for the two proteins in the processing of DNA lesions, as BRCA2 mutants contained more short deletions, with a wider size distribution, which frequently showed microhomology near the breakpoints resembling repair by non-homologous end joining. An increased and prolonged gamma-H2AX signal in MMS-treated BRCA1/2 cells suggested an aberrant processing of stalled replication forks as the cause of increased mutagenesis. The high rate of base substitution mutagenesis demonstrated by our experiments is likely to significantly contribute to the oncogenic effect of the inactivation of BRCA1 or BRCA2.

Correction: A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags.

Correction for 'A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags' by B. Söveges, et al., Org. Biomol. Chem., 2016, 14, 6071-6078.

A systematic study of protein labeling by fluorogenic probes using cysteine targeting vinyl sulfone-cyclooctyne tags.

Fluorescent tagging of proteins via accessible cysteine residues is of paramount importance. In this study, model proteins of interest (mitogen-activated protein kinases) were labeled successfully in native state on their free thiols by direct fluorescence derivatization, or in a sequential manner where conjugation of the site specific linker and the fluorophore is carried out in two steps. To this end we designed and prepared two novel chemical reporters carrying vinyl sulfone as Cys targeting function and cyclooctyne motifs, suitable for subsequent conjugation with fluorogenic azides via copper free strain-promoted azide-alkyne click chemistry. Direct and sequential labeling reaction steps were analyzed by native PAGE, capillary zone electrophoresis and tandem mass spectrometry. The efficiency of tagging was correlated with solvent accessibility of the Cys residues. Our results indicated that conjugation of native proteins by vinyl sulfone linkers was fast and thiol-selective. Subsequent click reaction with fluorogenic dyes generates intensive fluorescence signals and fulfills all requirements of bioorthogonality.

[Uterine fibroleiomyoma: a case of abdominal mass of extraordinary dimensions]. / Fibroleiomioma uterino: un caso di "massa addominale" di straordinarie dimensioni.

A case of abdominal mass strongly suspected at examination of being a primary ovarian tumour with abnormal echographic features is reported. At laparotomy, it proved to be a uterine fibroleiomyoma of extraordinary dimensions.

[Unusual case of biliary ileus]. / Un singolare caso di ileo biliare.

The clinical and radiological picture observed in a female patient with cholelithiasis urgently hospitalised for abdominal occlusion is described. Radiological examination of the digestive tract disclosed a bilio apico-bulbar fistula and ectasis and hypotonia of the jejuno-ileal loops. A large calculus composed of calcium salts and cholesterine buried in a sigmoid loop was spontaneously passed by the patient during the examination. This resulted in regression of the symptoms of pain and occlusion. Cholangiocholecystography and examination of the digestive tract 3 months later showed a normal gallbladder picture, and disappearance of the fistula.

[Radiological signs of a disease that is dying out: hepato-splenic atrophy and cholangiocarcinoma after administration of thorium dioxide (Thorotrast)]. / Segni radiologici di una patologia in via di estinzione: atrofia-epato-splenica e colangiocarcinoma dopo somministrazione di diossido di torio (Thorotrast).

The cases of two patients with radio-opaque residues of contrast medium (Thorotrast) in the abdomen, at the level of the liver, the spleen and certain lymph node groups of the hepatic hilum and the pancreas are reported. In one of the two patients (both of whom had undergone examination with this contrast medium in the '40s) necropsy showed the existence of cholangiocarcinoma. It is therefore considered useful to recall the X-ray signs typical of this pathology, which is dying out after the abandonment of thorium dioxide as an X-ray contrast medium.

[Clinical and radiological considerations on a case of acquired idiopathic agammaglobulinemia]. / Considerazioni cliniche e radiologiche in un caso di agammaglobulinemia idiopatica acquisita.

A case of acquired idiopathic agammaglobulinaemia in a 23-yr-old woman is presented. The picture included frequent and recurrent lung, sinus and mastoid infections, and diarrhoea. The pathogenesis, radiological picture, and differential diagnosis of this immunological disorder are described.