The spliceosome U2 snRNP factors promote genome stability through distinct mechanisms; transcription of repair factors and R-loop processing.

Recent whole-exome sequencing of malignancies have detected recurrent somatic mutations in U2 small nuclear ribonucleoprotein complex (snRNP) components of the spliceosome. These factors have also been identified as novel players in the DNA-damage response (DDR) in several genome-wide screens and proteomic analysis. Although accumulating evidence implies that the spliceosome has an important role in genome stability and is an emerging hallmark of cancer, its precise role in DNA repair still remains elusive. Here we identify two distinct mechanisms of how spliceosome U2 snRNP factors contribute to genome stability. We show that the spliceosome maintains protein levels of essential repair factors, thus contributing to homologous recombination repair. In addition, real-time laser microirradiation analysis identified rapid recruitment of the U2 snRNP factor SNRPA1 to DNA-damage sites. Functional analysis of SNRPA1 revealed a more immediate and direct role in preventing R-loop-induced DNA damage. Our present study implies a complex interrelation between transcription, mRNA splicing and the DDR. Cells require rapid spatio-temporal coordination of these chromatin transactions to cope with various forms of genotoxic stress.

Validation and development of MTH1 inhibitors for treatment of cancer.

BACKGROUND: Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. MATERIAL AND METHODS: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8-oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling, proteomics, cellular thermal shift assays, kinase and CEREP panel were used for target engagement, mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E-mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. RESULTS: Here, we demonstrate that recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579, (Karonudib), an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. CONCLUSION: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept.

The homeobox gene GBX2, a target of the myb oncogene, mediates autocrine growth and monocyte differentiation.

The homeobox gene GBX2 was identified as a target gene of the v-Myb oncoprotein encoded by the avian myeloblastosis virus (AMV). GBX2 activation by c-Myb requires signal transduction emanating from the cell surface while the leukemogenic AMV v-Myb constitutively induces the GBX2 gene. Mutations in the DNA binding domain of AMV-Myb render it independent of signaling events and concomitantly abrogate the collaboration between Myb and CCAAT Enhancer Binding Proteins (C/EBP), which are involved in granulocyte differentiation. Ectopic expression of GBX2 in growth factor-dependent myeloblasts induces monocytic features and independence from exogenous cytokines, reflecting distinct features of AMV-transformed cells. Our results suggest that Myb or factors it interacts with contribute to hematopoietic lineage choice and differentiation in a signal transduction-dependent fashion.

Densitometric analysis of lower molar interradicular areas in superposable radiographs.

Superposable radiographs of lower molar areas were obtained by means of a specially designed film holder and a gnathostat maintained in a constant position with respect to long-cone radiological equipement. A quantitative evaluation of the bone mass in the interradicular area was obtained by scanning the area under a photodensitometer. A photodensitometric scan of the image of an aluminium wedge adapted to each radiograph was also performed. This allowed the transformation, with the aid of a computer, of the photodensitometric recording of bone into a tracing of mm of aluminium equivalents. The reproducibility and precision of the method were verified by comparing magnifications of repeated radiographs, by photodensitometric readings of radiographs developed in different batches and by repeated quantitative readings of the same film. The method allowed one, for instance, to follow quantitatively the healing of a radiographically visible periapical lesion. It was also used to evaluate quantitatively, in the interradicular area, bone changes which could not be seen with the marked eye.

Clinical evaluation of four amalgam alloys: a five-year report.

In this study 96 teeth of 12 patients were restored with either the conventional alloy Premix, the blended non-gamma 2 amalgam Dispersalloy, or one of the spherical alloys Sybraloy and Tytin. To ascertain an objective comparison of the clinical performance of these alloys, two different alloys were used in each of at least two dental arch quadrants in the same patient and consequently were placed in the same oral environment. Clinical performance of the restorations was evaluated by macrophotography and scanning electron microphotography of replicas made after placement and 2, 3, and 5 years thereafter. Standard criteria were used for the evaluation of anatomic form, surface condition, and marginal adaptation of the fillings. In addition, the filling-enamel interface was assessed from the microphotographs. The three high-copper alloys performed better clinically than the conventional alloy, and the spherical alloys had the best qualifications. In addition to visual examination and photographic evaluation of restorations, the micrometric assessment of replica photographs from the scanning electron microscope may render clinical trials of amalgam alloys measurable and less subjective.

Mantle dentine in man--a quantitative microradiographic study.

50 microradiographs taken in a standardized manner of midsagittal ground sections of teeth of individuals aged 18 to 56 years were densitometrically evaluated along a track passing through enamel, dentine and an aluminium stepwedge. Semi-quantitative analysis of mineral density uniformly showed an irregular platform representing circumpulpal dentine and a peripheral down slope in the region of the amelodentinal junction, representing mantle dentine. The width of this less mineralized peripheral zone measured on densitometric recordings averaged 150 microns (+/- 50). Quantitative analysis of the two dentinal regions permitted the calculation of the mineral content in terms of volume percentage using both a graphic method and an electronic computer method. The sections were also examined by polarized light microscopy which clearly visualized the presence of peripheral mantle dentine. The mean mineral density of circumpulpal dentine was 46% according to both the graphic and the computer methods; mantle dentine yielded means close to 42% according by both methods. The 4% difference in density between circumpulpal dentine and mantle dentine proved to be statistically significant; there was no significant difference between the means obtained graphically and those obtained electronically. The need for further investigation of this region of the amelodentinal junction was stressed.

Attitude accessibility following a self-perception process.

Self-perception processes have been postulated to occur only to the extent that initial attitudes are weak. The present research asked whether the outcome of such a process is a strengthening of the attitude in question. Two experiments investigated the accessibility of attitudes from memory following self-inference from behavior. Experiment 1 examined the consequence for attitude accessibility of reviewing and considering previously performed religious behaviors that were recent and primarily unmanded versus distant in time and "manded" in nature. Experiment 2 involved the performance of a new behavior that was either required or freely chosen. In each case, control subjects either did not review prior behaviors or did not perform a new behavior. In both experiments, attitude accessibility, as measured by the latency of response to attitudinal inquiries, was enhanced by the consideration or performance of unmanded behavior, but not by manded behavior. The relevance of this finding to issues regarding attitude-behavior consistency and attitudinal persistence is discussed.