BRCA2 deficiency reveals that oxidative stress impairs RNaseH1 function to cripple mitochondrial DNA maintenance.

Oxidative stress is a ubiquitous cellular challenge implicated in aging, neurodegeneration, and cancer. By studying pathogenic mutations in the tumor suppressor BRCA2, we identify a general mechanism by which oxidative stress restricts mitochondrial (mt)DNA replication. BRCA2 inactivation induces R-loop accumulation in the mtDNA regulatory region and diminishes mtDNA replication initiation. In BRCA2-deficient cells, intracellular reactive oxygen species (ROS) are elevated, and ROS scavengers suppress the mtDNA defects. Conversely, wild-type cells exposed to oxidative stress by pharmacologic or genetic manipulation phenocopy these defects. Mechanistically, we find that 8-oxoguanine accumulation in mtDNA caused by oxidative stress suffices to impair recruitment of the mitochondrial enzyme RNaseH1 to sites of R-loop accrual, restricting mtDNA replication initiation. Thus, oxidative stress impairs RNaseH1 function to cripple mtDNA maintenance. Our findings highlight a molecular mechanism that links oxidative stress to mitochondrial dysfunction and is elicited by the inactivation of genes implicated in neurodegeneration and cancer.

Evaluating automated titre score as an alternative to continuous flow analysis for the prediction of passive anti-D in pregnancy.

OBJECTIVES: To evaluate the potential of the automated titre score (TS) as an alternative method to continuous flow analysis (CFA) for the prediction of the nature of anti-D in pregnancy. BACKGROUND: The 2016 revised British Society for Haematology (BSH) antenatal guidelines recommended a measurement of anti-D concentration by CFA to ensure the detection of potential immune anti-D. Due to high referral costs and resource pressures, uptake has been challenging for hospital laboratories. Serious Hazards of transfusion (SHOT) data have previously shown that this has contributed to missed antenatal follow ups for women with immune anti-D and neonates affected by haemolytic disease of the fetus/newborn. METHODS/MATERIALS: In this multicentre comparative study, samples referred for CFA quantification were also tested by an ORTHO VISION automated anti-D indirect antiglobulin test (IAT) serial dilution and then converted to TS. CFA results and history of anti-D prophylaxis were used to categorise samples as passive or immune, with the aim of determining a potential TS cut-off for CFA referral of at risk patients. RESULTS: Five UK National Health Service (NHS) trusts generated a total of 196 anti-D TS results, of which 128 were classified as passive and 68 as immune. Diagnostic testing of CFA and TS values indicated a TS cut-off of 35 to assist in distinguishing the nature of anti-D. Using this cut-off, 175 (89%) results were correctly assigned into the passive or immune range, giving a specificity of 92.19% and a negative predictive value of 91.47%. CONCLUSION: TS in conjunction with clinical and anti-D prophylaxis history can be used as a viable and cost-effective alternative to CFA in a hospital laboratory setting.

Association of high CD4-positive T cell infiltration with mutations in HLA class II-regulatory genes in microsatellite-unstable colorectal cancer.

Besides being expressed on professional antigen-presenting cells, HLA class II antigens are expressed on various tumors of non-lymphoid origin, including a subset of colorectal cancers (CRC). Information about the regulation of HLA class II antigen expression is important for a better understanding of their role in the interactions between tumor and immune cells. Whether lack of HLA class II antigen expression in tumors reflects the selective immune destruction of HLA class II antigen-expressing tumor cells is unknown. To address this question, we tested whether lack of HLA class II antigen expression in CRC was associated with immune cell infiltration. We selected microsatellite-unstable (MSI-H) CRC, because they show pronounced tumor antigen-specific immune responses and, in a subset of tumors, lack of HLA class II antigen expression due to mutations inactivating HLA class II-regulatory genes. We examined HLA class II antigen expression, mutations in regulatory genes, and CD4-positive T cell infiltration in 69 MSI-H CRC lesions. Mutations in RFX5, CIITA, and RFXAP were found in 13 (28.9%), 3 (6.7%), and 1 (2.2%) out of 45 HLA class II antigen-negative tumors. CD4-positive tumor-infiltrating lymphocyte counts were significantly higher in HLA class II antigen-negative tumors harboring mutations in HLA class II-regulatory genes (107.4 T cells per 0.25 mm(2)) compared to tumors without mutations (55.5 T cells per 0.25 mm(2), p = 0.008). Our results suggest that the outgrowth of tumor cells lacking HLA class II antigen expression due to mutations of regulatory genes is favored in an environment of dense CD4-positive T cell infiltration.

Expression of LY6D is induced at the surface of MCF10A cells by X-ray irradiation.

In order to identify membrane proteins whose expression is induced by X-ray irradiation, we developed an antibody (Ab)-directed strategy using a phage Ab library. X-Ray-irradiated cells were screened with a phage Ab library in the presence of a large excess of polyclonal Abs prepared against membrane proteins that are commonly present at the surface of both X-ray-irradiated and nonirradiated cells. After isolation of Ab that bound only to X-ray-irradiated cells, the antigen was identified using MS. Using this approach, we found that expression of LY6D is induced in MCF10A cells by X-ray irradiation. The induction of LY6D expression is triggered through a pathway regulated by ATM, CHK2 and p53. This method is a new Ab-directed proteomic strategy for analysis of membrane proteins, and is applicable to various biological phenomena in situations in which both target molecule-expressing cells and nonexpressing cells are available.

Deconstructing the late phase of vimentin assembly by total internal reflection fluorescence microscopy (TIRFM).

Quantitative imaging of intermediate filaments (IF) during the advanced phase of the assembly process is technically difficult, since the structures are several µm long and therefore they exceed the field of view of many electron (EM) or atomic force microscopy (AFM) techniques. Thereby quantitative studies become extremely laborious and time-consuming. To overcome these difficulties, we prepared fluorescently labeled vimentin for visualization by total internal reflection fluorescence microscopy (TIRFM). In order to investigate if the labeling influences the assembly properties of the protein, we first determined the association state of unlabeled vimentin mixed with increasing amounts of labeled vimentin under low ionic conditions by analytical ultracentrifugation. We found that bona fide tetrameric complexes were formed even when half of the vimentin was labeled. Moreover, we demonstrate by quantitative atomic force microscopy and electron microscopy that the morphology and the assembly properties of filaments were not affected when the fraction of labeled vimentin was below 10%. Using fast frame rates we observed the rapid deposition of fluorescently labeled IFs on glass supports by TIRFM in real time. By tracing their contours, we have calculated the persistence length of long immobilized vimentin IFs to 1 µm, a value that is identical to those determined for shorter unlabeled vimentin. These results indicate that the structural properties of the filaments were not affected significantly by the dye. Furthermore, in order to analyze the late elongation phase, we mixed long filaments containing either Alexa 488- or Alexa 647-labeled vimentin. The 'patchy' structure of the filaments obtained unambiguously showed the elongation of long IFs through direct end-to-end annealing of individual filaments.

Analyzing the regulation of metabolic pathways in human breast cancer.

BACKGROUND: Tumor therapy mainly attacks the metabolism to interfere the tumor's anabolism and signaling of proliferative second messengers. However, the metabolic demands of different cancers are very heterogeneous and depend on their origin of tissue, age, gender and other clinical parameters. We investigated tumor specific regulation in the metabolism of breast cancer. METHODS: For this, we mapped gene expression data from microarrays onto the corresponding enzymes and their metabolic reaction network. We used Haar Wavelet transforms on optimally arranged grid representations of metabolic pathways as a pattern recognition method to detect orchestrated regulation of neighboring enzymes in the network. Significant combined expression patterns were used to select metabolic pathways showing shifted regulation of the aggressive tumors. RESULTS: Besides up-regulation for energy production and nucleotide anabolism, we found an interesting cellular switch in the interplay of biosynthesis of steroids and bile acids. The biosynthesis of steroids was up-regulated for estrogen synthesis which is needed for proliferative signaling in breast cancer. In turn, the decomposition of steroid precursors was blocked by down-regulation of the bile acid pathway. CONCLUSION: We applied an intelligent pattern recognition method for analyzing the regulation of metabolism and elucidated substantial regulation of human breast cancer at the interplay of cholesterol biosynthesis and bile acid metabolism pointing to specific breast cancer treatment.