BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1 ^−/− mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1 ^−/− mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to γ-irradiation. BRIT1 ^−/− MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1 ^−/− mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2''s function and as a result leads to infertility and genomic instability in mice.     

NCI Workshop Report: Clinical and Computational Requirements for Correlating Imaging Phenotypes with Genomics Signatures

The National Cancer Institute (NCI) Cancer Imaging Program organized two related workshops on June 26–27, 2013, entitled “Correlating Imaging Phenotypes with Genomics Signatures Research” and “Scalable Computational Resources as Required for Imaging-Genomics Decision Support Systems.” The first workshop focused on clinical and scientific requirements, exploring our knowledge of phenotypic characteristics of cancer biological properties to determine whether the field is sufficiently advanced to correlate with imaging phenotypes that underpin genomics and clinical outcomes, and exploring new scientific methods to extract phenotypic features from medical images and relate them to genomics analyses. The second workshop focused on computational methods that explore informatics and computational requirements to extract phenotypic features from medical images and relate them to genomics analyses and improve the accessibility and speed of dissemination of existing NIH resources. These workshops linked clinical and scientific requirements of currently known phenotypic and genotypic cancer biology characteristics with imaging phenotypes that underpin genomics and clinical outcomes. The group generated a set of recommendations to NCI leadership and the research community that encourage and support development of the emerging radiogenomics research field to address short-and longer-term goals in cancer research.     

Sleep states alter ventral medullary surface responses to blood pressure challenges

Ventral medullary surface (VMS) activity declines during rapid eye movement (REM) sleep, suggesting a potential for reduced VMS responsiveness to blood pressure challenges during that state. We measured VMS neural activity, assessed as changes in reflected 660-nm wavelength light, during pressor and depressor challenges within sleep/waking states in five adult, unrestrained, unanesthetized cats and in two control cats. Phenylephrine elevated blood pressure and elicited an initial VMS activity decline and a subsequent rise in VMS activity in all states, although the initial decline during quiet sleep occurred only in rostral placements. Phasic REM periods elicited a momentary recovery from the evoked activity rise, and arousals diminished the overall elevation in activity. A sodium nitroprusside depressor challenge increased VMS activity more in REM sleep than in quiet sleep, with the increase being even less in waking. Enhanced responses to depressor challenges during REM sleep suggest a loss of dampening of evoked activity during that state; state-related differential baroreflex sensitivity may result from sleep-waking changes in VMS responses to blood pressure challenges.     

The non-peptidyl fungal metabolite L-783,281 activates TRK neurotrophin receptors

Neurotrophin binding to the extracellular surface of the Trk family of tyrosine kinase receptors leads to the activation of multiple signalling cascades, culminating in neuroregenerative effects, including neuronal survival and neurite outgrowth. Since neurotrophins themselves are not ideal drug candidates due to their poor pharmacokinetic behaviour and bioavailability, small molecule neurotrophin mimetics may be beneficial in treating a number of neurodegenerative disorders. The present study demonstrates that L-783,281, a non-peptidyl fungal metabolite, is capable of stimulating TrkA, B and C phosphorylation to various extents in CHO cells stably expressing human Trk receptors. L-783,281 also stimulated Trk phosphorylation in a number of rat and human primary neuronal cultures, whereas the highly similar compound, L-767,827, was without effect. Mechanistic studies utilizing transiently transfected PDGF/TrkA and TrkA/PDGF chimeras, demonstrated that L-783,281 is likely to interact with the intracellular domain of the TrkA receptor. Further investigations suggested that L-783,281 was nevertheless able to instigate receptor dimerization by binding in a non-covalent manner. Although the cytotoxicity of the compound was shown to preclude its effects in neuronal survival and neurite outgrowth assays, it is a prototype for a small molecule neurotrophin mimetic that activates Trk by interacting at a site different from the neurotrophin-binding site.     

Melanin content and MC1R function independently affect UVR-induced DNA damage in cultured human melanocytes

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)-induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR-induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR-induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss-of-function MC1R, regardless of their MC or EC, sustained more UVR-induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR-induced DNA damage in melanocytes.     

Biosecurity risk factors presented by international vessels: a statistical analysis

Invasive non-indigenous species are among the greatest threats to global biodiversity. Shipping is the principal vector for international dispersal of nonindigenous species, and shipping rates are increasing globally. The Australian government performs a range of regulatory actions to mitigate biosecurity risks associated with marine vessels, and in so doing has amassed a large volume of operational inspection data. This data can be used to quantitatively examine risk factors of vessels failing biosecurity procedures after arriving from international ports, the nature of biosecurity failures, and the types and seizure rates of biosecurity risk material (BRM). Classification trees with gradient boosting were used to assess characteristics that predict high risk vessels (n = 93,006) for carrying BRM, across 7 years of inspection data. Undeclared vessels and suspected irregular entry vessels posed the highest risk, but both were rare. Vessels that visit infrequently (<20 visits in 7 years) were common and had almost three times greater odds of failing inspection than vessels visiting frequently. On statistical analysis, yachts appeared to pose less risk than commercial vessels. In operational terms, a tentative profiled 20% fraction would contain 57% of genuine failures, and the concomitant non-screened group would contain 82% of passes. The most common reason for inspection failures was ballast water non-compliance (2.53%) and plant or insect detections (1.77%); biofouling was less common (0.13%) but testing for biofouling is not exhaustive. Invertebrate species comprised almost 90% of invasive organisms detected and seized from vessels failing biosecurity inspections. This study targets an entire transportation vector, which includes many pathways. Understanding the characteristics of transport vectors is pivotal to characterising the risk of biological invasions and applying adequate controls and prevention strategies. Our results show that biosecurity risk is not uniform on maritime pathways, so there is considerable scope for biosecurity regulators to impose risk-based intervention.     

Decrease in ovarian platelet-activating factor during ovulation in the gonadotropin-primed immature rat

Platelet-activating factor (PAF) is a biologically active phospholipid that is released locally during acute inflammatory reactions and tissue injury. Since there is evidence that the biochemical events of mammalian ovulation resemble an inflammatory reaction, the objective of this study was to determine whether ovarian levels of PAF change during ovulation. At 2-h intervals during the ovulatory process in gonadotropin-primed 25-day-old Wistar rats, the ovaries were extirpated, homogenized, and extracted for lipids. The extracts were subjected to thin-layer chromatography (TLC), and the portion of the silica gel that comigrated with PAF was re-extracted and assayed for PAF activity. The PAF was measured (in fmole equivalents of synthetic PAF) by a bioassay based on the capacity of aliquots of the extracts to release [3H]-serotonin from platelets isolated from whole blood of rabbits and prelabeled with [3H]-serotonin. The ovarian level of PAF decreased (p less than 0.01) by 36% from 6.67 +/- 0.77 to 4.27 +/- 0.45 fmoles/mg ovary by 2 h after treatment with human chorionic gonadotropin (hCG), and it declined another 14% by 4 h after hCG. The ovarian PAF remained at this reduced level for up to 24 h after hCG. The administration of indomethacin (5 mg/rat, s.c.) or epostane (5 mg/rat, s.c.) at 1 h after hCG prevented ovulation, but neither drug affected the decline in ovarian PAF. Preliminary tests showed that the lipid extracts from the ovaries also contained PAF inhibitor(s) that comigrated with PAF on the TLC plates. Similar to PAF, the lipid-soluble inhibitor(s) decreased (p less than 0.05) in the ovaries within 4 h after hCG treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

RNA binding specificity of a Drosophila snRNP protein that shares sequence homology with mammalian U1-A and U2-B" proteins.

We have characterized a recombinant Drosophila melanogaster RNA binding protein, D25, by virtue of its antigenic relationship to mammalian U1 and U2 small nuclear ribonucleoprotein (U snRNP) proteins. Sequence analysis revealed that D25 bears strong similarity to both the human U1 snRNP-A (U1-A) and U2 snRNP-B" (U2-B") proteins. However, at residues known to be critical for the RNA binding specificities of U1-A and U2-B" D25 sequence is more similar to U2-B". Using direct RNA binding assays D25 selected U1 RNA from either HeLa or Drosophila Kc cell total RNA. Furthermore, D25 bound U1 RNA when transfected into mammalian cells. Thus, D25 appears to be a Drosophila homolog of the mammalian U1-A protein, despite its sequence similarity to U2-B".