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Duplex sequencing (DuplexSeq) is an error-corrected next-generation sequencing (ecNGS) method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors by comparing grouped strand sequencing reads. The resulting background of less than one artifactual mutation per 107nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DuplexSeq-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues, a considerable advancement compared to currently usedin vivogene mutation assays.HIGHLIGHTSDuplexSeq is an ultra-accurate NGS technology that directly quantifies mutationsENU-dependent mutagenesis was detected 24 h post-exposure in proliferative tissuesMultiple tissues exhibited the canonical ENU mutation spectrum 7 d after exposureResults obtained with DuplexSeq were highly concordant between laboratoriesThe Rat-50 Mutagenesis Assay is promising for applications in genetic toxicology

Sunlight UV exposure produces DNA photoproducts in skin that are repaired solely by nucleotide excision repair in humans. A significant fraction of melanomas are thought to result from UV-induced DNA damage that escapes repair; however, little evidence is available about the functional capacity of normal human melanocytes, malignant melanoma cells, and metastatic melanoma cells to repair UV-induced photoproducts in DNA. In this study, we measured nucleotide excision repair in both normal melanocytes and a panel of melanoma cell lines. Our results show that in 11 of 12 melanoma cell lines tested, UV photoproduct repair occurred as efficiently as in primary melanocytes. Importantly, repair capacity was not affected by mutation in the N-RAS or B-RAF oncogenes, nor was a difference observed between a highly metastatic melanoma cell line (A375SM) or its parental line (A375P). Lastly, we found that although p53 status contributed to photoproduct removal efficiency, its role did not seem to be mediated by enhanced expression or activity of DNA binding protein DDB2. We concluded that melanoma cells retain capacity for nucleotide excision repair, the loss of which probably does not commonly contribute to melanoma progression. Cancer Res; 70(12); 4922–30. ©2010 AACR.

Supplementary Table 1 from Similar Nucleotide Excision Repair Capacity in Melanocytes and Melanoma Cells

Supplementary Figure Legends 1-6 from Similar Nucleotide Excision Repair Capacity in Melanocytes and Melanoma Cells

Supplementary Figures 1-6 from Similar Nucleotide Excision Repair Capacity in Melanocytes and Melanoma Cells

Supplementary Table 2 from Similar Nucleotide Excision Repair Capacity in Melanocytes and Melanoma Cells

Currently, approximately 80,000 chemicals are used in commerce. Most have little-to-no toxicity information. The U.S. Toxicology in the 21st Century (Tox21) program has conducted a battery of in vitro assays using a quantitative high-throughput screening (qHTS) platform to gain toxicity information on environmental chemicals. Due to technical challenges, standard methods for providing xenobiotic metabolism could not be applied to qHTS assays. To address this limitation, we screened the Tox21 10,000-compound (10K) library, with concentrations ranging from 2.8 nM to 92 µM, using a p53 beta-lactamase reporter gene assay (p53-bla) alone or with rat liver microsomes (RLM) or human liver microsomes (HLM) supplemented with NADPH, to identify compounds that induce p53 signaling after biotransformation. Two hundred and seventy-eight compounds were identified as active under any of these three conditions. Of these 278 compounds, 73 gave more potent responses in the p53-bla assay with RLM, and 2 were more potent in the p53-bla assay with HLM compared with the responses they generated in the p53-bla assay without microsomes. To confirm the role of metabolism in the differential responses, we re-tested these 75 compounds in the absence of NADPH or with heat-attenuated microsomes. Forty-four compounds treated with RLM, but none with HLM, became less potent under these conditions, confirming the role of RLM in metabolic activation. Further evidence of biotransformation was obtained by measuring the half-life of the parent compounds in the presence of microsomes. Together, the data support the use of RLM in qHTS for identifying chemicals requiring biotransformation to induce biological responses.

Error-corrected Duplex Sequencing (DuplexSeq) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DuplexSeq to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent,N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25-200 µM) and DNA was sequenced 48, 72, and 96 h post-exposure. A DuplexSeq mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A robust increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (R2=0.95). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DuplexSeq for characterizing chemical mutagenicity in both research and regulatory evaluation.

Black cohosh (BC; Actaea racemosa L.), a top-selling botanical dietary supplement, is marketed to women primarily to ameliorate a variety of gynecological symptoms. Due to widespread usage, limited safety information, and sporadic reports of hepatotoxicity, the Division of the National Toxicology Program (DNTP) initially evaluated BC extract in female rats and mice. Following administration of up to 1000 mg/kg/day BC extract by gavage for 90 days, dose-related increases in micronucleated peripheral blood erythrocytes were observed, along with a nonregenerative macrocytic anemia resembling megaloblastic anemia in humans. Because both micronuclei and megaloblastic anemia may signal disruption of folate metabolism, and inadequate folate levels in early pregnancy can adversely affect neurodevelopment, the DNTP conducted a pilot cross-sectional study comparing erythrocyte micronucleus frequencies, folate and B12 levels, and a variety of hematological and clinical chemistry parameters between women who used BC and BC-naïve women. Twenty-three women were enrolled in the BC-exposed group and 28 in the BC-naïve group. Use of any brand of BC-only supplement for at least three months was required for inclusion in the BC-exposed group. Supplements were analyzed for chemical composition to allow cross-product comparisons. All participants were healthy, with no known exposures (e.g., x-rays, certain medications) that could influence study endpoints. Findings revealed no increased micronucleus frequencies and no hematological abnormalities in women who used BC supplements. Although reassuring, a larger, prospective study with fewer confounders (e.g., BC product diversity and duration of use) providing greater power to detect subtle effects would increase confidence in these findings.

Black cohosh extract (BCE) is marketed to women as an alternative to hormone replacement therapy for alleviating menopausal symptoms. Previous studies by the National Toxicology Program revealed that BCE induced micronuclei (MN) and a nonregenerative macrocytic anemia in rats and mice, likely caused by disruption of the folate metabolism pathway. Additional work using TK6 cells showed that BCE induced aneugenicity by destabilizing microtubules. In the present study, BCE-induced MN were confirmed in TK6 and HepG2 cells. We then evaluated BCE-induced DNA damage using the comet assay at multiple time points (0.5–24 h). Following a 0.5-h exposure, BCE induced significant, concentration-dependent increases in %tail DNA in TK6 cells only. Although DNA damage decreased in TK6 cells over time, likely due to repair, small but statistically significant levels of DNA damage were observed after 2 and 4 h exposures to 250 µg/ml BCE. A G1/S arrest in TK6 cells exposed to 125 µg/ml BCE (24 h) was accompanied by apoptosis and increased expression of γH2A.X, p-Chk1, p-Chk2, p53, and p21. Conditioning TK6 cells to physiological levels of folic acid (120 nM) did not increase the sensitivity of cells to BCE-induced DNA damage. BCE did not alter global DNA methylation in TK6 and HepG2 cells cultured in standard medium. Our results suggest that BCE induces acute DNA strand breaks which are quickly repaired in TK6 cells, whereas DNA damage seen at 4 and 24 h may reflect apoptosis. The present study supports that BCE is genotoxic mainly by inducing MN with an aneugenic mode of action.

The National Toxicology Program tested two common radiofrequency radiation (RFR) modulations emitted by cellular telephones in a 2-year rodent cancer bioassay that included interim assessments of additional animals for genotoxicity endpoints. Male and female Hsd:Sprague Dawley SD rats and B6C3F1/N mice were exposed from Gestation day 5 or Postnatal day 35, respectively, to code division multiple access (CDMA) or global system for mobile modulations over 18 hr/day, at 10-min intervals, in reverberation chambers at specific absorption rates of 1.5, 3, or 6 W/kg (rats, 900 MHz) or 2.5, 5, or 10 W/kg (mice, 1,900 MHz). After 19 (rats) or 14 (mice) weeks of exposure, animals were examined for evidence of RFR-associated genotoxicity using two different measures. Using the alkaline (pH > 13) comet assay, DNA damage was assessed in cells from three brain regions, liver cells, and peripheral blood leukocytes; using the micronucleus assay, chromosomal damage was assessed in immature and mature peripheral blood erythrocytes. Results of the comet assay showed significant increases in DNA damage in the frontal cortex of male mice (both modulations), leukocytes of female mice (CDMA only), and hippocampus of male rats (CDMA only). Increases in DNA damage judged to be equivocal were observed in several other tissues of rats and mice. No significant increases in micronucleated red blood cells were observed in rats or mice. In conclusion, these results suggest that exposure to RFR is associated with an increase in DNA damage. Environ. Mol. Mutagen. 61:276-290, 2020. © 2019 Wiley Periodicals, Inc.

Black cohosh extract (BCE) is a popular botanical dietary supplement marketed to relieve symptoms of various gynecological ailments. Studies conducted by the National Toxicology Program (NTP) showed that BCE induces micronucleated erythrocytes in female rats and mice. Subsequently, the NTP showed that a variety of BCEs, including the sample that induced micronuclei (MN) in vivo ("NTP BCE") had a similar effect in human TK6 cells. Further testing with the MultiFlow® DNA Damage Assay revealed that TK6 cells exposed to NTP BCE, as well as a BCE reference material (BC XRM), exhibited a signature consistent with aneugenic activity in TK6 cells. Results from experiments reported herein confirmed these in vitro observations with NTP BCE and BC XRM. We extended these studies to include a novel test system, the MultiFlow Aneugen Molecular Mechanism Assay. For these experiments, TK6 cells were exposed to NTP BCE and BC XRM over a range of concentrations in the presence of fluorescent Taxol (488 Taxol). After 4 h, nuclei from lysed cells were stained with a nucleic acid dye and labeled with fluorescent antibodies against phospho-histone H3 (p-H3) and Ki-67. Whereas BCEs did not affect p-H3:Ki-67 ratios (a signature of aneugenic mitotic kinase inhibitors), 488 Taxol-associated fluorescence (a tubulin binder-sensitive endpoint) was affected. More specifically, 488 Taxol-associated fluorescence was reduced over the same concentration range that was previously observed to induce MN. These results provide direct evidence that BCEs destabilize microtubules in vitro, and this is the molecular mechanism responsible for the aneugenicity findings. Environ. Mol. Mutagen. 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Botanical dietary supplements are complex mixtures with numerous potential sources of variation along the supply chain from raw plant material to the market. Approaches for determining sufficient similarity (ie, complex mixture read-across) may be required to extrapolate efficacy or safety data from a tested sample to other products containing the botanical ingredient(s) of interest. In this work, screening-level approaches for generating both chemical and biological-response profiles were used to evaluate the similarity of black cohosh (Actaea racemosa) and Echinacea purpurea samples to well-characterized National Toxicology Program (NTP) test articles. Data from nontargeted chemical analyses and gene expression of toxicologically important hepatic receptor pathways (aryl hydrocarbon receptor [AhR], constitutive androstane receptor [CAR], pregnane X receptor [PXR], farnesoid X receptor [FXR], and peroxisome proliferator-activated receptor alpha [PPARα]) in primary human hepatocyte cultures were used to determine similarity through hierarchical clustering. Although there were differences in chemical profiles across black cohosh samples, these differences were not reflected in the biological-response profiles. These findings highlight the complexity of biological-response dynamics that may not be reflected in chemical composition profiles. Thus, biological-response data could be used as the primary basis for determining similarity among black cohosh samples. Samples of E. purpurea displayed better correlation in similarity across chemical and biological-response measures. The general approaches described herein can be applied to complex mixtures with unidentified active constituents to determine when data from a tested mixture (eg, NTP test article) can be used for hazard identification of sufficiently similar mixtures, with the knowledge of toxicological targets informing assay selection when possible.

Genotoxicity is a critical component of a comprehensive toxicological profile. The Tox21 Program used five quantitative high throughput screening (qHTS) assays measuring some aspect of DNA damage/repair to provide information on the genotoxic potential of over 10,000 compounds. Included were assays detecting activation of p53, increases in the DNA repair protein ATAD5, phosphorylation of H2AX, and enhanced cytotoxicity in DT40 cells deficient in DNA repair proteins REV3 or KU70/RAD54. Each assay measures a distinct component of the DNA damage response signaling network; >70% of active compounds were detected in only one of the five assays. When qHTS results were compared to results from three standard genotoxicity assays (bacterial mutation, in vitro chromosomal aberration, and in vivo micronucleus), a maximum of 40% of known, direct-acting genotoxicants were active in one or more of the qHTS genotoxicity assays, indicating low sensitivity. This suggests that these qHTS assays cannot in their current form be used to replace traditional genotoxicity assays. However, despite the low sensitivity, ranking chemicals by potency of response in the qHTS assays revealed an enrichment for genotoxicants up to 12-fold compared with random selection, when allowing a 1% false positive rate. This finding indicates these qHTS assays can be used to prioritize chemicals for further investigation, allowing resources to focus on compounds most likely to induce genotoxic effects. To refine this prioritization process, models for predicting genotoxicity potential of chemicals that were active in Tox21 genotoxicity assays were constructed using all Tox21 assay data, yielding a prediction accuracy up to 0.83. Data from qHTS assays related to stress response pathway signaling (including genotoxicity) were the most informative for model construction. By using the results from qHTS genotoxicity assays, predictions from models based on qHTS data, and predictions from commercial bacterial mutagenicity QSAR models, we prioritized Tox21 chemicals for genotoxicity characterization.

Observations of the collisional evolution of particle ensembles in a microgravity environment are necessary to characterize the processes that lead to the formation of planetesimals, km-size and larger bodies, within the protoplanetary disk. The two current theories of planetesimal formation, namely growth through binary sticking collisions and gravitational instability within the protoplanetary disk, have difficulties in explaining how particles grow beyond a centimeter in size. In this paper we describe the CubeSat Particle Aggregation and Collision Experiment (Q-PACE), a Low Earth Orbit 3U CubeSat mission that will provide a high-quality, long duration microgravity environment in which we will observe collisions between particles under conditions relevant to planetesimal formation. We have designed a series of experiments involving a broad range of particle size, density, surface properties, and collision velocities to observe collisional outcomes from bouncing to sticking as well as aggregate disruption in tens of thousands of collisions.

Black cohosh extract (BCE) is a widely used dietary supplement marketed to women to alleviate symptoms of gynecological ailments, yet its toxicity has not been well characterized. The National Toxicology Program (NTP) previously reported significant increases in micronucleated erythrocytes in peripheral blood of female Wistar Han rats and B6C3F1/N mice administered 15-1,000 mg BCE/kg/day by gavage for 90 days. These animals also developed a dose-dependent nonregenerative macrocytic anemia characterized by clinical changes consistent with megaloblastic anemia. Both micronuclei (MN) and megaloblastic anemia can arise from disruption of the folate metabolism pathway. The NTP used in vitro approaches to investigate whether the NTP's test lot of BCE, BCEs from various suppliers, and root powders from BC and other cohosh species, were genotoxic in general, and to gain insight into the mechanism of action of BCE genotoxicity. Samples were tested in human TK6 lymphoblastoid cells using the In Vitro MicroFlow® MN assay. The NTP BCE and a BC extract reference material (XRM) were tested in the MultiFlow® DNA Damage assay, which assesses biomarkers of DNA damage, cell division, and cytotoxicity. The NTP BCE and several additional BCEs were tested in bacterial mutagenicity assays. All samples induced MN when cells were grown in physiological levels of folic acid. The NTP BCE and BC XRM produced activity patterns consistent with an aneugenic mode of action. The NTP BCE and five additional BCEs were negative in bacterial mutagenicity tests. These findings show that black cohosh preparations induce chromosomal damage and may pose a safety concern. Environ. Mol. Mutagen. 59:416-426, 2018. © 2018 Published 2018. This article is a US Government work and is in the public domain in the USA.

Genotoxicity potential is a critical component of any comprehensive toxicological profile. Compounds that induce DNA or chromosomal damage often activate p53, a transcription factor essential to cell cycle regulation. Thus, within the US Tox21 Program, we screened a library of ∼10,000 (∼8,300 unique) environmental compounds and drugs for activation of the p53-signaling pathway using a quantitative high-throughput screening assay employing HCT-116 cells (p53+/+ ) containing a stably integrated β-lactamase reporter gene under control of the p53 response element (p53RE). Cells were exposed (-S9) for 16 hr at 15 concentrations (generally 1.2 nM to 92 μM) three times, independently. Excluding compounds that failed analytical chemistry analysis or were suspected of inducing assay interference, 365 (4.7%) of 7,849 unique compounds were concluded to activate p53. As part of an in-depth characterization of our results, we first compared them with results from traditional in vitro genotoxicity assays (bacterial mutation, chromosomal aberration); ∼15% of known, direct-acting genotoxicants in our library activated the p53RE. Mining the Comparative Toxicogenomics Database revealed that these p53 actives were significantly associated with increased expression of p53 downstream genes involved in DNA damage responses. Furthermore, 53 chemical substructures associated with genotoxicity were enriched in certain classes of p53 actives, for example, anthracyclines (antineoplastics) and vinca alkaloids (tubulin disruptors). Interestingly, the tubulin disruptors manifested unusual nonmonotonic concentration response curves suggesting activity through a unique p53 regulatory mechanism. Through the analysis of our results, we aim to define a role for this assay as one component of a comprehensive toxicological characterization of large compound libraries. Environ. Mol. Mutagen. 58:494-507, 2017. © 2017 Wiley Periodicals, Inc.

Methods for quantifying DNA damage, as well as repair of that damage, in a high-throughput format are lacking. Single cell gel electrophoresis (SCGE; comet assay) is a widely-used method due to its technical simplicity and sensitivity, but the standard comet assay has limitations in reproducibility and throughput. We have advanced the SCGE assay by creating a 96-well hardware platform coupled with dedicated data processing software (CometChip Platform). Based on the original cometchip approach, the CometChip Platform increases capacity ~200 times over the traditional slide-based SCGE protocol, with excellent reproducibility. We tested this platform in several applications, demonstrating a broad range of potential uses including the routine identification of DNA damaging agents, using a 74-compound library provided by the National Toxicology Program. Additionally, we demonstrated how this tool can be used to evaluate human populations by analysis of peripheral blood mononuclear cells to characterize susceptibility to genotoxic exposures, with implications for epidemiological studies. In summary, we demonstrated a high level of reproducibility and quantitative capacity for the CometChip Platform, making it suitable for high-throughput screening to identify and characterize genotoxic agents in large compound libraries, as well as for human epidemiological studies of genetic diversity relating to DNA damage and repair., National Institutes of Health (U.S.) (Grant CA148629), National Institutes of Health (U.S.) (Grant ES021116)

Rodent lung tumors are morphologically similar to a subtype of human lung adenocarcinomas. The objective of this study was to evaluate Kirsten rat sarcoma oncogene homolog ( Kras), epidermal growth factor receptor ( Egfr), and tumor protein 53 ( Tp53) mutations, which are relevant to human lung cancer, in cobalt metal dust (CMD)-induced alveolar/bronchiolar tumors of B6C3F1/N mice and F344/NTac rats. Kras mutations were detected in 67% (mice) and 31% (rats) of CMD-induced lung tumors and were predominantly exon 1 codon 12 G to T transversions (80% in mice and 57% in rats). Egfr mutations were detected in 17% (both mice and rats) of CMD-induced lung tumors and were predominantly in exon 20 with 50% G to A transitions (mice and rats). Tp53 mutations were detected in 19% (mice) and 23% (rats) of CMD-induced lung tumors and were predominant in exon 5 (mice, 69% transversions) and exon 6 (rats, all transitions). No mutations were observed for these genes in spontaneous lung tumors or normal lungs from untreated controls. Ames assay indicated that CMD is mutagenic in the absence but not in the presence of S9 mix. Thus, the mutation data (G to T transversions) and Ames assay results suggest that oxidative damage to DNA may be a contributing factor in CMD-induced pulmonary carcinogenesis in rodents.

// Stephanie L. Smith-Roe 1, 5 , Jun Nakamura 2 , Darcy Holley 1 , Paul D. Chastain II 3, 4 , Gary B. Rosson 1 , Dennis A. Simpson 3 , John R. Ridpath 3 , David G. Kaufman 3 , William K. Kaufmann 3 , Scott J. Bultman 1 1 Department of Genetics and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA 2 Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC, USA 3 Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC, USA 4 Department of Biomedical Sciences, William Carey University, Hattiesburg, MS, USA 5 Current address : Division of the National Toxicology Program, NIEHS, Research Triangle Park, NC, USA Correspondence to: Scott J. Bultman, e-mail: Scott_Bultman@med.unc.edu Keywords: BRG1, BRM, tumor suppression, DNA damage response, chemotherapeutics Received: September 29, 2014 Accepted: November 09, 2014 Published: January 07, 2015 ABSTRACT SWI/SNF complexes utilize BRG1 (also known as SMARCA4) or BRM (also known as SMARCA2) as alternative catalytic subunits with ATPase activity to remodel chromatin. These chromatin-remodeling complexes are required for mammalian development and are mutated in ~20% of all human primary tumors. Yet our knowledge of their tumor-suppressor mechanism is limited. To investigate the role of SWI/SNF complexes in the DNA-damage response (DDR), we used shRNAs to deplete BRG1 and BRM and then exposed these cells to a panel of 6 genotoxic agents. Compared to controls, the shRNA knockdown cells were hypersensitive to certain genotoxic agents that cause double-strand breaks (DSBs) associated with stalled/collapsed replication forks but not to ionizing radiation-induced DSBs that arise independently of DNA replication. These findings were supported by our analysis of DDR kinases, which demonstrated a more prominent role for SWI/SNF in the activation of the ATR-Chk1 pathway than the ATM-Chk2 pathway. Surprisingly, γH2AX induction was attenuated in shRNA knockdown cells exposed to a topoisomerase II inhibitor (etoposide) but not to other genotoxic agents including IR. However, this finding is compatible with recent studies linking SWI/SNF with TOP2A and TOP2BP1. Depletion of BRG1 and BRM did not result in genomic instability in a tumor-derived cell line but did result in nucleoplasmic bridges in normal human fibroblasts. Taken together, these results suggest that SWI/SNF tumor-suppressor activity involves a role in the DDR to attenuate replicative stress and genomic instability. These results may also help to inform the selection of chemotherapeutics for tumors deficient for SWI/SNF function.

This paper updates on the recent development of the novel Solid State Inflation Balloon (SSIB), a simple, reliable, low-cost, non-propulsive deorbit mechanism for the full range of small satellites, defined by NASA as less than 180 kg. It aims to focus on the recent demonstration, for the first time, inflation of a ∼10 cm sized balloon in a vacuum chamber. Small satellites typically rely on aerodynamic drag to deorbit within the FAA’s 25 year requirements. The SSIB will enhance aerodynamic drag by inflating a balloon at the end-of-life of a satellite mission. This technology will provide a scalable and non-existing capability, low-cost deorbit, for applications in the full-range of smallsats, from CubeSats to MicroSats. The SSIB system is composed of three major components: a Micro-Electro-Mechanical Systems (MEMS) Solid-State Gas Generator (SSGG) chip, a balloon structure made of thin film compatible with space environment (i.e. Mylar, Kapton, or Teflon), and a sub-system package suitable for spacecraft integration. The SSGG is composed of a 2D addressable array of sodium azide (NaN3) crystals, confined by Su-8 wells, on a glass substrate. Current versions include 2×2 and 8×8 arrays designed for a full range of small satellites. Under each well is a resistive heater and when heated to above 350 °C, the NaN3 spontaneously decomposes to generate N2 gas in time scales on the order of 10 milliseconds. Each well is designed with a typical volume of 10–15 m3 to 10−6 m3 of NaN3 (i.e. 1,500 μm × 1,500 μm × 150 μm on the larger end of the spectrum). The SSIB system is low power (∼1 W per well for less than 10 seconds) and have low mass (∼100 grams, where mass is dominated by the size of the required balloon). Initial simulations have shown that the SSIB with balloons of 1 m2 cross-section can deorbit small satellites from above 1000 km well within 25 years.

The in vitro MultiFlow DNA Damage assay multiplexes p53, γH2AX, phospho-histone H3, and polyploidization biomarkers into 1 flow cytometric analysis (Bryce, S. M., Bernacki, D. T., Bemis, J. C., and Dertinger, S. D. (2016). Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach. Environ. Mol. Mutagen. 57, 171-189). The work reported herein evaluated the generalizability of the method, as well as several data analytics strategies, to a range of chemical classes not studied previously. TK6 cells were exposed to each of 103 diverse chemicals, 86 of which were supplied by the National Toxicology Program (NTP) and selected based upon responses in genetic damage assays conducted under the Tox21 program. Exposures occurred for 24 h over a range of concentrations, and cell aliquots were removed at 4 and 24 h for analysis. Multiplexed response data were evaluated using 3 machine learning models designed to predict genotoxic mode of action based on data from a training set of 85 previously studied chemicals. Of 54 chemicals with sufficient information to make an a priori call on genotoxic potential, the prediction models' accuracies were 79.6% (random forest), 88.9% (logistic regression), and 90.7% (artificial neural network). A majority vote ensemble of the 3 models provided 92.6% accuracy. Forty-nine NTP chemicals were not adequately tested (maximum concentration did not approach assay's cytotoxicity limit) and/or had insufficient conventional genotoxicity data to allow their genotoxic potential to be defined. For these chemicals MultiFlow data will be useful in future research and hypothesis testing. Collectively, the results suggest the MultiFlow assay and associated data analysis strategies are broadly generalizable, demonstrating high predictability when applied to new chemicals and classes of compounds.

We present the first MEMS Solid State Gas Generator (SSGG) for use as a deorbit technology, in conjunction with inflation balloons, for Small-Satellites ( 3 ) crystals on a glass substrate. The crystals are contained in wells formed by a thick-film of epoxy (SU-8). Under each well is a resistive heater that is selectively addressed using Metal-Insulator-Metal (MIM) diode networks. When heated to above 350 °C, the NaN 3 spontaneously decomposes to generate the N 2 gas in time scales on the order of 10 milliseconds. Each well can be designed with a typical volume of 10−15 m3 to 10−6 m3 of NaN 3 . The effectiveness of the system has been numerically evaluated for 1U, 2U and 3U CubeSats. The deorbit time for these three spacecraft from a 550km altitude has been calculated to be 118, 236, and 355 days respectively, independent of initial inclination.

As a result of measurements acquired by the Cassini–Huygens mission of Titan’s near surface atmospheric composition and temperature, Titan conditions can now be simulated in the laboratory and samples can subsequently be subjected to those conditions. Titan demonstrates an active hydrological-like cycle with its thick atmosphere, dynamic clouds, polar lakes of methane and ethane, moist regolith, and extensive fluvial erosive features. Unlike Earth, Titan’s hydrological-like cycle likely involves several constituents, primarily methane and ethane. Here the properties of a new Titan simulation facility are presented, including conceptual methodology, design, implementation, and performance results. The chamber maintains Titan’s surface temperature and pressure, and the sample cryogenic liquids undergoing experimentation are condensed within the chamber itself. During the experiments, the evaporation rates of the sample liquids are directly determined by continually measuring mass. Constituents are analyzed utilizing a Fourier Transform Infrared Spectroscopy (FTIR), and vapor concentrations are determined using a gas chromatograph fitted with a Flame Ionization Detector (FID). All pertinent data is logged via computer. Under laboratory conditions, the direct measurements of the evaporation rates of methane, ethane, and mixtures thereof can be achieved. Among the processes to be studied are the effects of regolith on transport from the subsurface to the atmosphere, the freezing point depression effects of dissolved nitrogen, and the solubility of various relevant organic compounds.

In late 2012, the members of the Environmental Mutagen Society voted to change its name to the Environmental Mutagenesis and Genomics Society. Here, we describe the thought process that led to adoption of the new name, which both respects the rich history of a Society founded in 1969 and reflects the many advances in our understanding of the nature and breadth of gene-environment interactions during the intervening 43 years.

Genomic instability is a hallmark of breast cancer, contributes to tumor heterogeneity, and influences chemotherapy resistance. Although Gap 2 and mitotic checkpoints are thought to prevent genomic instability, the role of these checkpoints in breast cancer is poorly understood. Here, we assess the Gap 2 and mitotic checkpoint functions of 24 breast cancer and immortalized mammary epithelial cell lines representing four of the six intrinsic molecular subtypes of breast cancer. We found that patterns of cell cycle checkpoint deregulation were associated with the intrinsic molecular subtype of breast cancer cell lines. Specifically, the luminal B and basal-like cell lines harbored two molecularly distinct Gap 2/mitosis checkpoint defects (impairment of the decatenation Gap 2 checkpoint and the spindle assembly checkpoint, respectively). All subtypes of breast cancer cell lines examined displayed aberrant DNA synthesis/Gap 2/mitosis progression and the basal-like and claudin-low cell lines exhibited increased percentages of chromatid cohesion defects. Furthermore, a decatenation Gap 2 checkpoint gene expression signature identified in the cell line panel correlated with clinical outcomes in breast cancer patients, suggesting that breast tumors may also harbor defects in decatenation Gap 2 checkpoint function. Taken together, these data imply that pharmacological targeting of signaling pathways driving these phenotypes may lead to the development of novel personalized treatment strategies for the latter two subtypes which currently lack targeted therapeutic options because of their triple negative breast cancer status., Cell cycle: Breast cancer subtypes exhibit distinct checkpoint dysfunctions Unique subtypes of breast cancer display distinctive patterns of genomic instability that affect cell division. Jacquelyn J. Bower and colleagues at the University of North Carolina at Chapel Hill, USA, assessed the molecular function of several G2-M cell cycle checkpoints—which ensure that cells don’t begin mitotic cell division until DNA replication is complete and they have had a chance to repair any damaged DNA—in 24 breast cancer and mammary epithelial cell lines that collectively represent four of the six intrinsic molecular subtypes of breast cancer. The distinct patterns of cell cycle checkpoint deregulation that the researchers found suggest that pharmacological targeting of the dysfunction in each type of breast cancer could lead to personalized treatment strategies. The findings also point to diagnostic biomarkers that could help predict patient responses to novel drugs or existing chemotherapies.

Acetylene is thought to be abundant on Titan according to most photochemical models. While detected in the atmosphere, its likely presence at the surface still lacks physical evidence. It is thought that solid acetylene could be a major component of Titan’s lakes shorelines and dry lakebed, detected as the 5 μm-bright deposits with the Cassini/VIMS instrument. Acetylene could also be present under its liquid form as dissolved solids in Titan’s methane–ethane lakes, as emphasized by thermodynamics studies. This paper is devoted to the near-infrared spectroscopy study of acetylene under solid and liquid phases between 1 and 2.2 μm, synthesized in a Titan simulation chamber that is able to reproduce extreme temperature conditions. From experiments, we observed a ∼10% albedo increase between liquid acetylene at 193–188 K and solid acetylene at 93 K. Using the NIR spectroscopy technique we successfully calculated the reflectivity ratio of solid/liquid acetylene as 1.13. The second difference we observed between liquid and solid acetylene is a shift in the major absorption band detected at 1.54 μm, the shift of ∼0.01 μm occurring toward higher wavelength. In order to assess the detectability of acetylene on Titan using the Cassini/VIMS instrument, we adapted our spectra to the VIMS spectral resolution. The spectral band at 1.55 μm and a negative slope at 2.0 μm falls in the Cassini/VIMS atmospheric windows over several VIMS infrared spectels, thus Cassini/VIMS should be able to detect acetylene.

The Optical Probe for Regolith Analysis (OPRA) is a spike-shaped subsurface analytical probe that will be delivered to a planet, asteroid, or cometary body by a lander and/or rover. OPRA will be pushed down into the subsurface to record near infrared spectra as a function of depth down to maximum of 50 cm. Therefore, knowledge of the required penetration force to specific depths can be helpful in estimating the length of the probe. Test probes covering the anticipated diameter (2.5, 1.9, 1.2 and 0.9 cm diameter) and tip angle (T.A. = 30°, 60°, 90° and 120°) of OPRA were inserted mechanically into dry playground sand. The results showed that tip angle does not have a major effect, while probe diameter and density of the regolith are the most important parameters. Increasing probe diameter from 0.9 to 1.9 cm (i.e. a factor of 2) leads to an increase in penetration force from 200 to 1000 N (i.e. a factor of 5) at 20 cm depth. An increase in bulk density (B.D.) from 1550 to 1700 kg m −3 leads to an increase in penetration force from 10 to 200 N at 20 cm depth. Bearing capacity theory was used to explain the downward movement of the penetrometer through regolith and showed good agreement with the experimental results. This model was then used to take into account the effect of gravity on other planetary bodies. We observed a good agreement between the theoretical model and results from penetration testings on the Moon by the Apollo missions. Since the maximum allowed force for penetration is the weight of the lander/rover on their targeted planetary surface, our results put a strong constraint on the maximum reachable depth without endangering the whole mission.

Dynamic models of the martian polar caps are in abundance, but most rely on the assumption that the rate of sublimation of CO 2 ice can be calculated from heat transfer and lack experimental verification. We experimentally measured the sublimation rate of pure CO 2 ice under simulated martian conditions as a test of this assumption, developed a model based on our experimental results, and compared our model's predictions with observations from several martian missions (MRO, MGS, Viking). We show that sun irradiance is the primary control for the sublimation of CO 2 ice on the martian poles with the amount of radiation penetrating the surface being controlled by variations in the optical depth, ensuring the formation and sublimation of the seasonal cap. Our model confirmed by comparison of MGS-MOC and MRO-HiRISE images, separated by 2–3 martian years, shows that ∼0.4 m are currently being lost from the south perennial cap per martian year. At this rate, the ∼2.4-m-thick south CO 2 perennial cap will disappear in about 6–7 martian years, unless a short-scale climatic cycle alters this rate of retreat.

Gullies are widespread on slopes on the surface of Mars and have been investigated by numerous authors, yet their formation processes remain elusive. In an attempt to understand the possibility of a water-based origin for these forms, we undertook a series of flume experiments at Earth surface temperatures and pressures. Our objectives were to produce forms that resemble those most commonly observed on Mars, documenting their morphometric characteristics and identifying any statistically significant relationships between form and controlling factors of slope and flow rate. Experiments were conducted in a 1×1.5 m2 flume filled with medium grain size sand. The experiments were run over a slope angle range of 10–30°, corresponding to the range for gullies on Mars. Water from a constant-head tank fed through 5 mm silicone hose to a rotameter and then released just below the surface at the top of the slope. Gullies were produced at slope angle values of 10°, 20°, and 30° and flow rate values of 445, 705, 965, and 1260 mL min−1 at each angle. Eighteen parameters were identified and subsequently measured on each gully produced in the flume. Gully forms were successfully reproduced and displayed development of the fundamental morphological components observed on Mars: alcove, channel, and apron. Slope–gully form relationships for each component revealed the following results: higher slope angles formed shorter gullies with thicker apron deposits. Moreover, longer gullies were seen at higher flow rates. We concluded that forms visually similar to those observed on Mars can be created by water in the laboratory flume under terrestrial conditions. Morphometric parameters can be measured and permit identification of controlling factors. Experimental simulation of gullies appears possible with proper scaling of experimental parameters. Although not directly scalable to Mars, flume gully parameters may be used to develop numerical models in the future.

We have studied the effect of wind velocity on the sublimation rate of pure water ice under martian conditions. Measurements were made for wind velocities ranging from 0.7 to 11.4 m s −1 , a typical range observed by the meteorological instruments on the surface of Mars, and at − 15 ° C a value typical of the daily high temperature for most of the year at the Pathfinder landing site. At this temperature, and for a low-humidity environment (relative humidity around 1%) sublimation rates increase following a linear trend of equation E S = 0.68 + 0.025 V ( E S is the sublimation rate in mm h −1 and V is the wind speed in m s −1 ). In high relative humidity (30–35%) atmospheres, the effect of wind velocity is negligible, and the sublimation rate remains nearly constant at 0.33 ± 0.04 mm h −1 . Pure forced convection theory did not provide a satisfying description of the data in terms of the range of values and their wind speed dependency. Therefore, a new semi-empirical expression for the sublimation rate that combines free and forced convection was developed using analogy with heat transfer models. Using this expression, sublimation rates of ice as a function of wind velocity for any temperature can be calculated. In general, temperature is more important that wind speed and atmospheric humidity in determining the rate of sublimation of ice on Mars.

Disruption of the DNA mismatch repair (MMR) pathway results in elevated mutation rates, inappropriate survival of cells bearing DNA damage, and increased cancer risk. Relatively little is known about the potential impact of environmentally relevant carcinogens on cancer risk in individuals with MMR-deficiency. We determined the effect of MMR status ( Mlh1 +/+ versus Mlh1 −/− ) on mutagenesis induced by the cooked-meat mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) within cII and supFG1 transgene reporters. Despite being a lymphomagen in mice, PhIP was not mutagenic in thymus. In colon, PhIP exposure induced 3-fold more mutations in Mlh1-deficient mice compared to their Mlh1 +/+ littermates. Similar induction was seen in Mlh1 −/− small intestine. Analysis of mutational spectra revealed that G/C to T/A transversions, the “signature PhIP mutation”, were induced to similar levels regardless of Mlh1 status. In contrast, Mlh1 −/− mice exhibited hypermutability to frameshifts, G/C to A/T transitions, and G/C to C/G transversions. Thus, both the level and types of mutation induced by PhIP are influenced by the activity of the MMR system. MMR may suppress PhIP-induced mutation through recognition and processing of specific mispairs (PhIP-G/T, PhIP-G/G, and PhIP-G/loop mispairs). In contrast, the PhIP-G/A mispair is unlikely to be a MMR substrate. In addition, the similar induction of both transversions and transitions in Mlh1 −/− mice suggests that mutagenic bypass of PhIP-G is similarly efficient with dATP, dTTP, and dGTP, in contrast to previously published conclusions. Our data suggests that MMR-deficiency would increase the likelihood of PhIP-induced carcinogenic mutations. Further evaluation of the risk that consumption of heterocyclic amines may impart to MMR-deficient individuals therefore is warranted. © 2005 Elsevier B.V. All rights reserved.

Optical emission spectroscopy (OES), absorption measurements, and thermal energy rate analysis were used in tandem with numerical models to characterize microwave argon plasmas. A WAVEMAT (model MPDR-3135) microwave diamond deposition system was used to generate argon plasmas at 5 Torr. Three excited state number densities (4p, 5p, and 5d) were obtained from the OES measurements, and a fourth excited state number density (4s) was obtained from the absorption measurements. Further, power absorbed in the substrate was monitored. A self-consistent two-dimensional argon model coupled with an electromagnetic field model and a 25-level two-dimensional (2D)-collisional-radiative model (CRM) was developed and validated with the experimental measurements. The 2D model provides the gas and electron temperature distributions, and the electron, ion, and 4s state number densities, which are then iteratively fed into the electromagnetic and CRM models. Both the numerically predicted thermal energy rates and excited sta...

Book Review: Chemical Dynamics in Extreme Environments, Rainer A. Dressler (Ed.). World Scientific Publishing Company, Singapore (2001).

The recently established Arkansas–Oklahoma Center for Space and Planetary Science has been given a large planetary simulation chamber by the Jet Propulsion Laboratory, Pasadena, California. When completely refurbished, the chamber will be dubbed Andromeda and it will enable conditions in space, on asteroids, on comet nuclei, and on Mars, to be reproduced on the meter-scale and surface and subsurface processes monitored using a range of analytical instruments. The following projects are currently planned for the facility. (1) Examination of the role of surface and subsurface processes on small bodies in the formation of meteorites. (2) Development of in situ sediment dating instrumentation for Mars. (3) Studies of the survivability of methanogenic microorganisms under conditions resembling the subsurface of Mars to test the feasibility of such species surviving on Mars and identify the characteristics of the species most likely to be present on Mars. (4) The nature of the biochemical “fingerprints” likely to have been left by live organisms on Mars from a study of degradation products of biologically related molecules. (5) Testing local resource utilization in spacecraft design. (6) Characterization of surface effects on reflectivity spectra for comparison with the data from spacecraft-borne instruments on Mars orbiters.

Inflatable and other membrane structures are expected to become increasingly important in space exploration due to their light weight and low cost. Unlike rigid structures, these structures are typically fabricated of flexible polymers and require internal pressurization to achieve structural integrity. Due to this, inflatable structures are vulnerable to the harsh space environment and catastrophic failure from structural vibration. A MEMS-based health monitoring and control system (HMCS) for space inflatables has been developed at the University of Arkansas. Fabricated mostly from polymeric materials, the HMCS is lightweight, flexible and can be attached directly to the external surface of an inflatable to provide health monitoring. Structure-wise, the HMCS is a three-dimensional multichip module with a sensor layer at the top, a common polyimide substrate in the middle and an actuator layer at the bottom. The sensor layer consists of an interconnected network of MEMS sensors for monitoring the environmental conditions around the inflatable and also the structural vibration of the inflatable. The actuator layer, fabricated from electroactive polymers, provides a two-dimensional shape control capability to the HMCS. When operated with strain and vibration sensors in the sensor layer, the polymer actuator can deform the surface contour of the inflatable to remove `wrinkles' and dampen structural vibration.

A drag-measurement facility was used to evaluate potential scramjet fuel-injection/wall-cooling cone gurations. Mach-2aire owcontainingatrainofweak,unsteady,ree ectingshockswassuppliedtotestplatesequippedwithforce and static-pressureinstrumentation. Anonporouse atplate,apartially porouse atplate,anda wallcavityplate (the latter two with mass injection ) were tested. The nonporous plate results were within 10% of both theoretical and numerical predictions. Theporousplateexhibited reduced drag (relative to thenonporous e at plate ), even without mass addition, caused by pressure equalization across the impinging shocks. Addition of mass decreased the drag 46%relativetothee at-platevalue.Therectangularwallcavityhadageometricaspectratio (lengthine owdirection to depth)of 5.0. The cavity plate produced thehighest drag of the cases tested. Without secondary injection cavityplate drag exceeded the instrumentation range, but was estimated as higher than the e at plate by approximately one order of magnitude. A minimum amount of secondary e ow injected into the cavity reduced the drag to a value 50% higher than the e at plate. Further cavity mass addition increased the drag. The porous e at plate provided drag reduction, whereas the cavity cone guration demonstrated drag penalties when compared to the e at plate.

These experiments addressed the role of striatal N-methyl-D-aspartate (NMDA) receptors in spatial behavior in the radial arm maze. Rats treated with the NMDA antagonist D-2-amino-5-phosphonopentanoic acid (AP-5) in the nucleus accumbens core, medial caudate, and posterior caudate were all significantly impaired in acquiring the correct spatial responses. In contrast, rats infused with AP-5 in the nucleus accumbens shell showed little impairment. When rats in all groups had learned the maze and were performing at similar levels, AP-5 had relatively little effect except in the posterior caudate group, where errors and trial times were again increased. These findings demonstrate the importance of NMDA receptor-dependent activity within the accumbens and caudate in spatial learning and performance. The neural processes necessary for adaptive spatial learning in complex environments may recruit multiple cortical systems having specialized functions, which in turn are integrated in widespread striatal regions.

Helium was injected normally to a Mach 3 airstream to simulate hydrogen fuel injection in a scramjet combustor. Two geometries were evaluated, a wedge-shaped wall orie ce and a circular wall orie ce. Injection was sonic in both geometries, and the expansion ratios and mass e ow rates were matched to isolate the effects of the geometric difference. Surface oil e ow patterns were inspected to determine the extent of boundary-layer separation upstream of each injector, shadowgraphs were used to visualize the e owe elds, and probe measurements were utilized to determine local helium concentrations. The wedgeshaped injection scheme demonstrated more rapid penetration into the freestream and increased mixing when compared to the baseline circular orie ce. In addition, the oil e ow photography showed that the wedge-shaped injector had no upstream separation zone, whereas the circular injector had a large separation zone. The wedge cone guration would therefore be expected to exhibit reduced wall heat transfer in an actual combustor. It is concluded that wedge-shaped, normal, fuel injectors should provide generally better performance than circular normal injectors in supersonic combustors.

In late 2012, the members of the Environmental Mutagen Society voted to change its name to the Environmental Mutagenesis and Genomics Society. Here, we describe the thought process that led to adoption of the new name, which both respects the rich history of a Society founded in 1969 and reflects the many advances in our understanding of the nature and breadth of gene-environment interactions during the intervening 43 years.

The ATR-dependent intra-S checkpoint protects DNA replication forks undergoing replication stress. The checkpoint is enforced by ATR-dependent phosphorylation of CHK1, which is mediated by the TIMELESS-TIPIN complex and CLASPIN. Although loss of checkpoint proteins is associated with spontaneous chromosomal instability, few studies have examined the contribution of these proteins to unchallenged DNA metabolism in human cells that have not undergone carcinogenesis or crisis. Furthermore, the TIMELESS-TIPIN complex and CLASPIN may promote replication fork protection independently of CHK1 activation. Normal human fibroblasts (NHF) were depleted of ATR, CHK1, TIMELESS, TIPIN or CLASPIN and chromosomal aberrations, DNA synthesis, activation of the DNA damage response (DDR) and clonogenic survival were evaluated. This work demonstrates in NHF lines from two individuals that ATR and CHK1 promote chromosomal stability by different mechanisms that depletion of CHK1 produces phenotypes that resemble more closely the depletion of TIPIN or CLASPIN than the depletion of ATR, and that TIMELESS has a distinct contribution to suppression of chromosomal instability that is independent of its heterodimeric partner, TIPIN. Therefore, ATR, CHK1, TIMELESS-TIPIN and CLASPIN have functions for preservation of intrinsic chromosomal stability that are separate from their cooperation for activation of the intra-S checkpoint response to experimentally induced replication stress. These data reveal a complex and coordinated program of genome maintenance enforced by proteins known for their intra-S checkpoint function.

Mammalian SWI/SNF complexes utilize either BRG1 or BRM as alternative catalytic subunits with DNA-dependent ATPase activity to remodel chromatin. Although the two proteins are 75 % identical, broadly expressed, and have similar biochemical activities in vitro, BRG1 is essential for mouse embryonic development, while BRM is dispensable. To investigate whether BRG1 and BRM have overlapping functions during mouse embryogenesis, we performed double-heterozygous intercrosses using constitutive null mutations previously created by gene targeting. The progeny of these crosses had a distribution of genotypes that was significantly skewed relative to their combined gene dosage. This was most pronounced at the top and bottom of the gene dosage hierarchy, with a 1.5-fold overrepresentation of Brg1 (+/+) ;Brm (+/+) mice and a corresponding 1.6-fold underrepresentation of Brg1 (+/-) ;Brm (-/-) mice. To account for the underrepresentation of Brg1 (+/-) ;Brm (-/-) mice, timed matings and blastocyst outgrowth assays demonstrated that ~50 % of these embryos failed to develop beyond the peri-implantation stage. These results challenge the idea that BRG1 is the exclusive catalytic subunit of SWI/SNF complexes in ES cells and suggest that BRM also interacts with the pluripotency transcription factors to facilitate self-renewal of the inner cell mass. In contrast to implantation, the Brm genotype did not influence an exencephaly phenotype that arises because of Brg1 haploinsufficiency during neural tube closure and that results in peri-natal lethality. Taken together, these results support the idea that BRG1 and BRM have overlapping functions for certain developmental processes but not others during embryogenesis.