Co-transcriptional R-loops are the main cause of estrogen-induced DNA damage.

The hormone estrogen (E2) binds the estrogen receptor to promote transcription of E2-responsive genes in the breast and other tissues. E2 also has links to genomic instability, and elevated E2 levels are tied to breast cancer. Here, we show that E2 stimulation causes a rapid, global increase in the formation of R-loops, co-transcriptional RNA-DNA products, which in some instances have been linked to DNA damage. We show that E2-dependent R-loop formation and breast cancer rearrangements are highly enriched at E2-responsive genomic loci and that E2 induces DNA replication-dependent double-strand breaks (DSBs). Strikingly, many DSBs that accumulate in response to E2 are R-loop dependent. Thus, R-loops resulting from the E2 transcriptional response are a significant source of DNA damage. This work reveals a novel mechanism by which E2 stimulation leads to genomic instability and highlights how transcriptional programs play an important role in shaping the genomic landscape of DNA damage susceptibility

Natural Organic Matter Characterization and Lead Speciation as Indicators of Potential Lead Bioavailability in Aquatic Environments

Trace metals and natural organic matter (NOM) are ubiquitous in aquatic environments. Some trace metals are essential for aquatic life, while other non-essential metals (like lead) can be toxic if present in great enough concentrations. Natural waters contain a combination of inorganic, and organic ligands capable of binding metals. While the chemistry of inorganic Pb species is well understood, and National Institute of Standards and Technology (NIST) certified K values are readily available, the interactions between metals and organic matter is not so clearly defined. It is important to understand how NOM interacts with metals in the environment, as the properties of NOM vary with source. If these differences in NOM chemistry induce source-dependent Pb binding, the industrial and environmental implications would be significant. This study aimed to characterize a variety of NOM sources of diverse origin, measure Pb speciation in order to determine if Pb-NOM binding is indeed source-dependent, and to determine which property/ies would best explain Pb-NOM binding. NOM sources were characterized using: total organic carbon/dissolved organic carbon (TOC/DOC), fluorescence excitation-emission matrices (FEEM), parallel factor analysis (PARAFAC), fluorescence index (FI), specific absorption coefficient (SAC340), chromium-reducible sulfide (CRS), thiol, dissolved organic nitrogen (DON), and proton binding index (PBI). These methods allowed for a quantification of organic carbon; humic acid-, fulvic acid-, tyrosine-, and tryptophan-like components; origin; aromaticity; sulfide ligands; nitrogen ligands; and oxygen ligands. SAC340, FI, %HA, %FA, %Trp, %Tyr, CRS, thiol, DON, and PBI values ranged from: 7.76 – 40.84, 1.04 – 1.84, 46.41 – 82.41%, 13.32 – 39.21%, 1.02 – 16.21%, 1.34 – 14.99%, 2.03 – 89.0 nmol/mgC, 71.8 – 186.5 nmol/mgC, 35.76 – 253.8 μgN/mgC, and 0.33 – 1.72 respectively. No one parameter, or simple series of parameters was able to discriminate NOM source. However, CRS, Trp, and Tyr may be able to discriminate saltwater from freshwater sources, while SAC340, CRS, thiol, DON, Trp, Tyr, and PBI may be able to discriminate between freshwater sources. Sources of terrestrial origin had significant SAC340 and PBI, while sources of microbial origin had significant CRS, DON, Trp, and Tyr. Free lead was then measured using flow-through titrations with a commercially available Pb ion-selective electrode (ISE) and an internal calibration method. To confirm that this method was applicable for trace-level analysis in NOM, ethylenediamine (EN) was used as a model ligand in both artificial freshwater (AFW) and artificial seawater (ASW), and the speciation modelled using certified logK values from NIST. In both AFW and ASW, the ISE accurately and reproducibly (within a factor of two) measured Pb2+ speciation with EN as a model ligand. However, when this method was applied to speciation measurement in NOM, measured values did not agree well with WHAM. This indicates that the fundamental assumption (that Pb-NOM binding will not occur at low pH) made by the internal calibration method is not effective at predicting speciation in NOM, as WHAM predicts that Pb-NOM binding will occur at low pH. An alternate calibration method was tested – forcing measured values to agree with WHAM at low pH – and gave much better agreement. However, speciation measurements in NOM demonstrated reproducible variation with source(indicating source-dependent Pb-NOM binding) which was not described by WHAM within a factor of two, regardless of the calibration method. DON and SAC340 for the titrated sources are significantly different, and may potentially explain differences in source-dependent Pb speciation in freshwater environments. This is of immense industrial and environmental importance, as current water quality guidelines (WQG) do not account for NOM, DON, or SAC340. Consequently, current guidelines could overestimate toxicity in highly aromatic sources, or underestimate toxicity in sources with high DON

River Run Red: The Fort Pillow Massacre in the American Civil War

Civil War Atrocity Nathan B. Forrest raid on Fort Pillow Andrew Ward is a professional writer whose previous works included Fits and Starts: The Premature Memoirs of Andrew Ward; The Blood Seed: A Novel of India; Our Bones Are Scattered: The Cawnpore Massacres in the Indian ...

Correlated fragile site expression allows the identification of candidate fragile genes involved in immunity and associated with carcinogenesis

Common fragile sites (cfs) are specific regions in the human genome that are particularly prone to genomic instability under conditions of replicative stress. Several investigations support the view that common fragile sites play a role in carcinogenesis. We discuss a genome-wide approach based on graph theory and Gene Ontology vocabulary for the functional characterization of common fragile sites and for the identification of genes that contribute to tumour cell biology. CFS were assembled in a network based on a simple measure of correlation among common fragile site patterns of expression. By applying robust measurements to capture in quantitative terms the non triviality of the network, we identified several topological features clearly indicating departure from the Erdos-Renyi random graph model. The most important outcome was the presence of an unexpected large connected component far below the percolation threshold. Most of the best characterized common fragile sites belonged to this connected component. By filtering this connected component with Gene Ontology, statistically significant shared functional features were detected. Common fragile sites were found to be enriched for genes associated to the immune response and to mechanisms involved in tumour progression such as extracellular space remodeling and angiogenesis. Our results support the hypothesis that fragile sites serve a function; we propose that fragility is linked to a coordinated regulation of fragile genes expression.Comment: 18 pages, accepted for publication in BMC Bioinformatic

The Civil War and the Limits of Destruction

The Destructive War? Mark E. Neely, Jr., McCabe-Greer Professor of the History of the Civil War Era at Pennsylvania State University, has written numerous books, primarily on political, cultural, and legal aspects of the period. As a senior figure in the field, he has unquestionably read e...

The Mre11-Rad50-Nbs1 complex mediates activation of TopBP1 by ATM

The activation of ATR-ATRIP in response to double-stranded DNA breaks (DSBs) depends upon ATM in human cells and Xenopus egg extracts. One important aspect of this dependency involves regulation of TopBP1 by ATM. In Xenopus egg extracts, ATM associates with TopBP1 and thereupon phosphorylates it on S1131. This phosphorylation enhances the capacity of TopBP1 to activate the ATR-ATRIP complex. We show that TopBP1 also interacts with the Mre11-Rad50-Nbs1 (MRN) complex in egg extracts in a checkpoint-regulated manner. This interaction involves the Nbs1 subunit of the complex. ATM can no longer interact with TopBP1 in Nbs1-depleted egg extracts, which suggests that the MRN complex helps to bridge ATM and TopBP1 together. The association between TopBP1 and Nbs1 involves the first pair of BRCT repeats in TopBP1. In addition, the two tandem BRCT repeats of Nbs1 are required for this binding. Functional studies with mutated forms of TopBP1 and Nbs1 suggested that the BRCT-dependent association of these proteins is critical for a normal checkpoint response to DSBs. These findings suggest that the MRN complex is a crucial mediator in the process whereby ATM promotes the TopBP1-dependent activation of ATR-ATRIP in response to DSBs

Congenital microcephaly

The underlying etiologies of genetic congenital microcephaly are complex and multifactorial. Recently, with the exponential growth in the identification and characterization of novel genetic causes of congenital microcephaly, there has been a consolidation and emergence of certain themes concerning underlying pathomechanisms. These include abnormal mitotic microtubule spindle structure, numerical and structural abnormalities of the centrosome, altered cilia function, impaired DNA repair, DNA Damage Response signaling and DNA replication, along with attenuated cell cycle checkpoint proficiency. Many of these processes are highly interconnected. Interestingly, a defect in a gene whose encoded protein has a canonical function in one of these processes can often have multiple impacts at the cellular level involving several of these pathways. Here, we overview the key pathomechanistic themes underlying profound congenital microcephaly, and emphasize their interconnected nature

An essential function for the ATR-Activation-Domain (AAD) of TopBP1 in mouse development and cellular senescence

ATR activation is dependent on temporal and spatial interactions with partner proteins. In the budding yeast model, three proteins – Dpb11TopBP1, Ddc1Rad9 and Dna2 - all interact with and activate Mec1ATR. Each contains an ATR activation domain (ADD) that interacts directly with the Mec1ATR:Ddc2ATRIP complex. Any of the Dpb11TopBP1, Ddc1Rad9 or Dna2 ADDs is sufficient to activate Mec1ATR in vitro. All three can also independently activate Mec1ATR in vivo: the checkpoint is lost only when all three AADs are absent. In metazoans, only TopBP1 has been identified as a direct ATR activator. Depletion-replacement approaches suggest the TopBP1-AAD is both sufficient and necessary for ATR activation. The physiological function of the TopBP1 AAD is, however, unknown. We created a knock-in point mutation (W1147R) that ablates mouse TopBP1-AAD function. TopBP1-W1147R is early embryonic lethal. To analyse TopBP1-W1147R cellular function in vivo, we silenced the wild type TopBP1 allele in heterozygous MEFs. AAD inactivation impaired cell proliferation, promoted premature senescence and compromised Chk1 signalling following UV irradiation. We also show enforced TopBP1 dimerization promotes ATR-dependent Chk1 phosphorylation. Our data suggest that, unlike the yeast models, the TopBP1-AAD is the major activator of ATR, sustaining cell proliferation and embryonic development