Wildfire Impact on Indoor and Outdoor PAH Air Quality.

Air quality impacts from wildfires are poorly understood, particularly indoors. As frequencies increase, it is important to optimize methodologies to understand and reduce chemical exposures from wildfires. Public health recommendations use air quality estimates from outdoor stationary air monitors, discounting indoor air conditions, and do not consider chemicals in the vapor phase, known to elicit adverse effects. We investigated vapor-phase polycyclic aromatic hydrocarbons (PAHs) in indoor and outdoor air before, during, and after wildfires using a community-engaged research approach. Paired passive air samplers were deployed at 15 locations across four states. Twelve unique PAHs were detected only in outdoor air during wildfires, highlighting a PAH exposure mixture for future study. Heavy-molecular-weight (HMW) outdoor PAH concentrations and average Air Quality Index (AQI) values were positively correlated (p < 0.001). Indoor PAH concentrations were higher in 77% of samples across all sampling events. Even during wildfires, 58% of sampled locations still had higher indoor PAH air concentrations. When AQI values exceeded 140 (unhealthy for sensitive groups), outdoor PAH concentrations became similar to or higher than indoors. Cancer and noncancer inhalation risk estimates from vapor-phase PAHs were higher indoors than outdoors, regardless of the wildfire impact. Consideration of indoor air quality and vapor-phase PAHs could inform public health recommendations regarding wildfires.

Elevational differences in trait response to UV-B radiation by long-toed salamander populations.

Amphibian species capable of optimizing trait response to environmental stressors may develop complex strategies for defending against rapid environmental change. Trait responses may differ between populations, particularly if stressor strength varies across spatial or temporal gradients. Ultraviolet-B (UV-B) radiation is one such stressor that poses a significant threat to amphibian species. We examined the ability of long-toed salamanders (Ambystoma macrodactylum) at high- and low-elevation breeding sites to cooperatively employ behavioral and physiological trait responses to mediate UV-B damage. We performed a microhabitat survey to examine differences in oviposition behavior and UV-B conditions among breeding populations at high- (n = 3; >1,500 m) and low-elevation (n = 3; <100 m) sites. We found significant differences in oviposition behavior across populations, with females at high-elevation sites selecting oviposition substrates in UV-B protected microhabitats. We also collected eggs (n = 633) from each of the breeding sites for analysis of photolyase activity, a photoreactivating enzyme that repairs UV-B damage to the DNA, using a photoproduct immunoassay. Our results revealed no significant differences in photolyase activity between long-toed salamander populations at high and low elevations. For high-elevation salamander populations, relatively low physiological repair capabilities in embryos appear to be buffered by extensive behavioral modifications to reduce UV-B exposure and standardize developmental temperatures. This study provides valuable insight into environmental stress responses via the assessment of multiple traits in allowing sensitive species to persist in rapidly changing landscapes.

Concurrent assessment of diffusive and advective PAH movement strongly affected by temporal and spatial changes.

Chemical movement influences exposure, remediation and interventions. Understanding chemical movement in addition to chemical concentrations at contaminated sites is critical to informed decision making. Using seepage meters and passive sampling devices we assessed both diffusive and advective flux of bioavailable polycyclic aromatic hydrocarbons (PAHs) at three time points, across two seasons, at a former creosote site in St. Helens, Oregon, United States. To our knowledge, this is the first time both diffusive and advective fluxes have been measured simultaneously at a contaminated site. Concentrations of 39 parent PAHs were determined by gas chromatography triple quadrupole mass spectrometry. Across both seasons and all sites, diffusive flux of PAHs was up to three orders of magnitude larger than advective flux. Release of PAHs from sediments and water were identified, likely from legacy contamination, as well as deposition from the air into the site from contemporary and other sources. The majority of PAH movement was comprised of three and four ring PAHs. Chemical movement on the site was found to be spatially and temporally variable. Volatilization decreased and atmospheric deposition increased from summer to fall. At the locations with higher levels of contamination, sum PAH release from sediments decreased by more than two orders of magnitude from summer to late fall. These data reflect the spatial heterogeneity and temporal variability of this site and demonstrate the importance of seasonality in assessing chemical movement at contaminated sites. Results from this study can inform future legacy site assessments to optimize remediation strategies and assess remediation effectiveness.

Reversion-reporter transgenes to analyze all six base-substitution pathways in Arabidopsis.

To expand the repertoire of Arabidopsis (Arabidopsis thaliana) mutation-reporter transgenes, we constructed six mutant alleles in the same codon of the ß-glucuronidase-encoding GUS transgene. Each allele reverts to GUS+ only via a particular one of the six transition/transversion pathways. AcV5 epitope tags, fused carboxyl terminal to the inactive GUS- proteins, enabled semiquantitative immunoassays in plant protein extracts. Spontaneous G:C→T:A transversions, previously not measured using reporter transgenes, were quite frequent. This may reflect mispairing of adenine with 8-oxoguanine in DNA attacked by endogenous oxyradicals. Spontaneous G:C→A:T was modest and other reversions were relatively low, as reported previously. Frequencies of ultraviolet C-induced TT→TC and TC→TT reversions were both high. With increased transgene copy number, spontaneous G:C→T:A reversions increased but ultraviolet C-induced reversions decreased. Frequencies of some reversion events were reduced among T4 versus T3 generation plants. Based on these and other analyses of sources of experimental variation, we propose guidelines for the employment of these lines to study genotoxic stress in planta.

Associating Increased Chemical Exposure to Hurricane Harvey in a Longitudinal Panel Using Silicone Wristbands.

Hurricane Harvey was associated with flood-related damage to chemical plants and oil refineries, and the flooding of hazardous waste sites, including 13 Superfund sites. As clean-up efforts began, concerns were raised regarding the human health impact of possible increased chemical exposure resulting from the hurricane and subsequent flooding. Personal sampling devices in the form of silicone wristbands were deployed to a longitudinal panel of individuals (n = 99) within 45 days of the hurricane and again one year later in the Houston metropolitan area. Using gas chromatography−mass spectroscopy, each wristband was screened for 1500 chemicals and analyzed for 63 polycyclic aromatic hydrocarbons (PAHs). Chemical exposure levels found on the wristbands were generally higher post-Hurricane Harvey. In the 1500 screen, 188 chemicals were detected, 29 were detected in at least 30% of the study population, and of those, 79% (n = 23) were found in significantly higher concentrations (p < 0.05) post-Hurricane Harvey. Similarly, in PAH analysis, 51 chemicals were detected, 31 were detected in at least 30% of the study population, and 39% (n = 12) were found at statistically higher concentrations (p < 0.05) post-Hurricane Harvey. This study indicates that there were increased levels of chemical exposure after Hurricane Harvey in the Houston metropolitan area.

Preschool-Age Children's Pesticide Exposures in Child Care Centers and at Home in Northern California.

INTRODUCTION: Young children may be exposed to pesticides used in child care centers and their family homes. We examined pesticide use and environmental and behavioral factors potentially associated with child exposures in these settings. METHOD: Preschool-age children (n = 125) wore silicone wristbands to assess pesticide exposures in their child care centers and home environments. Information about environmental and behavioral exposure determinants was collected using parent surveys, child care director interviews, and observations. RESULTS: Commonly detected pesticides were bifenthrin, chlorpyrifos, cypermethrin, fipronil, and cis- and trans-permethrin. Pesticide chemical storage onsite, cracks in the walls, using doormats, observed pests, or evidence of pests were associated with child exposures. Exposures were higher in counties with higher agricultural or commercial pesticide use or when children lived in homes near agricultural fields. DISCUSSION: Young children are being exposed to harmful pesticides, and interventions are needed to lower their risk of health problems later in life.

Firefighter exposures to potential endocrine disrupting chemicals measured by military-style silicone dog tags.

Studies suggest that exposure to potential endocrine disrupting chemicals (pEDCs) may contribute to adverse health outcomes, but pEDC exposures among firefighters have not been fully characterized. Previously, we demonstrated the military-style silicone dog tag as a personal passive sampling device for assessing polycyclic aromatic hydrocarbon exposures among structural firefighters. This follow-up analysis examined the pEDC exposures based on department call volume, duty shift, and questionnaire variables. Structural firefighters (n = 56) were from one high and one low fire call volume department (Kansas City, MO metropolitan area) and wore separate dog tags while on- and off-duty (ndogtags = 110). The targeted 1530 analyte semi-quantitative screening method was conducted using gas chromatography mass spectrometry (npEDCs = 433). A total of 47 pEDCs were detected, and several less-frequently-detected pEDCs (<75%) were more commonly detected in off- compared to on-duty dog tags (conditional logistic regression). Of the 11 phthalates and fragrances detected most frequently (>75%), off-duty pEDC concentrations were strongly correlated (r = 0.31-0.82, p < 0.05), suggesting co-applications of phthalates and fragrances in consumer products. Questionnaire variables of "regular use of conventional cleaning products" and "fireplace in the home" were associated with select elevated pEDC concentrations by duty shift (paired t-test). This suggested researchers should include detailed questions about consumer product use and home environment when examining personal pEDC exposures.

Discovery of firefighter chemical exposures using military-style silicone dog tags.

Occupational chemical hazards in the fire service are hypothesized to play a role in increased cancer risk, and reliable sampling technologies are necessary for conducting firefighter chemical exposure assessments. This study presents the military-style dog tag as a new configuration of silicone passive sampling device to sample individual firefighters' exposures at one high and one low fire call volume department in the Kansas City, Missouri metropolitan area. The recruited firefighters (n = 56) wore separate dog tags to assess on- and off-duty exposures (ndogtags = 110), for a total of 30 24 h shifts. Using a 63 PAH method (GC-MS/MS), the tags detected 45 unique PAHs, of which 18 have not been previously reported as firefighting exposures. PAH concentrations were higher for on- compared to off-duty tags (0.25 < Cohen's d ≤ 0.80) and for the high compared to the low fire call volume department (0.25 ≤ d < 0.70). Using a 1530 analyte screening method (GC-MS), di-n-butyl phthalate, diisobutyl phthalate, guaiacol, and DEET were commonly detected analytes. The number of fire attacks a firefighter participated in was more strongly correlated with PAH concentrations than firefighter rank or years in the fire service. This suggested that quantitative data should be employed for firefighter exposure assessments, rather than surrogate measures. Because several detected analytes are listed as possible carcinogens, future firefighter exposure studies should consider evaluating complex mixtures to assess individual health risks.

Reciprocal chromosome translocation associated with TDNA-insertion mutation in Arabidopsis: genetic and cytological analyses of consequences for gametophyte development and for construction of doubly mutant lines.

Chromosomal rearrangements may complicate construction of Arabidopsis with multiple TDNA-insertion mutations. Here, crossing two lines homozygous for insertions in AtREV3 and AtPOLH (chromosomes I and V, respectively) and selfing F1 plants yielded non-Mendelian F2 genotype distributions: frequencies of +/++/+ and 1/1 2/2 progeny were only 0.42 and 0.25%. However, the normal development and fertility of double mutants showed AtPOLH-1 and AtREV3-2 gametes and 1/1 2/2 embryos to be fully viable. F2 distributions could be quantitatively predicted by assuming that F1 selfing produced inviable (1,2) and (+,+) gametophytes 86% of the time. Some defect intrinsic to the F1 selfing process itself thus appeared responsible. In selfing AtREV3 (+/2 ) single mutants, imaging of ovules and pollen showed arrest or abortion, respectively, of half of gametophytes; however, gametogenesis was normal in AtREV3 ( 2/2 ) homozygotes. These findings, taken together, suggested that T-DNA insertion at AtREV3 on chromosome I had caused a reciprocal I-V translocation. Spreads of meiosis I chromosomes in selfing AtREV3 (+/2 ) heterozygotes revealed the predicted cruciform four-chromosome structures, which fluorescence in situ hybridization showed to invariably include both translocated and normal chromosomes I and V. Sequencing of the two junctions of T-DNA with AtREV3 DNA and the two with gene At5g59920 suggested translocation via homologous recombination between independent inverted-repeat T-DNA insertions. Thus, when crosses between TDNA-insertion mutants yield anomalous progeny distributions, TDNA-linked translocations should be considered.

Artificial turf: chemical flux and development of silicone wristband partitioning coefficients.

This work provides the first quantitative measure of in situ flux of semi-volatile contaminants on artificial turf fields. Passive samplers were used to identify gas-phase PAHs and OPAHs not previously reported associated with artificial turf. Utilizing a broad and targeted screen, we assess both artificial turf and from crumb rubber for 1,529 chemicals, including several with known health effects including benzo[c]fluorene. We also report the presence of 25 chemicals that have not yet been reported in artificial turf literature, including some with known effects on human health. This is the first report of bioavailable gas-phase PAH and OPAH concentrations on an outdoor field, to date gas-phase concentrations have only been reported from indoor facilities. Turf air and air were highly correlated at all three sites, and particularly at the recently-installed indoor site. Finally, thermal extraction and silicone passive samplers are highly suitable for larger-scale sampling campaigns that aim for less solvent and sample processing. We demonstrate for the first time that silicone passive samplers can be used to quantify volatile and semi-volatile organic chemicals from artificial turf. Co-deploying silicone passive samplers and conventional low density polyethylene, we develop partitioning coefficients that can be used for silicone passive air sampling environmental assessment.

Preparation and performance features of wristband samplers and considerations for chemical exposure assessment.

Wristbands are increasingly used for assessing personal chemical exposures. Unlike some exposure assessment tools, guidelines for wristbands, such as preparation, applicable chemicals, and transport and storage logistics, are lacking. We tested the wristband's capacity to capture and retain 148 chemicals including polychlorinated biphenyls (PCBs), pesticides, flame retardants, polycyclic aromatic hydrocarbons (PAHs), and volatile organic chemicals (VOCs). The chemicals span a wide range of physical-chemical properties, with log octanol-air partitioning coefficients from 2.1 to 13.7. All chemicals were quantitatively and precisely recovered from initial exposures, averaging 102% recovery with relative SD ≤21%. In simulated transport conditions at +30 °C, SVOCs were stable up to 1 month (average: 104%) and VOC levels were unchanged (average: 99%) for 7 days. During long-term storage at -20 °C up to 3 (VOCs) or 6 months (SVOCs), all chemical levels were stable from chemical degradation or diffusional losses, averaging 110%. Applying a paired wristband/active sampler study with human participants, the first estimates of wristband-air partitioning coefficients for PAHs are presented to aid in environmental air concentration estimates. Extrapolation of these stability results to other chemicals within the same physical-chemical parameters is expected to yield similar results. As we better define wristband characteristics, wristbands can be better integrated in exposure science and epidemiological studies.

Discrimination and versatility in mismatch repair.

Evolutionarily-conserved mismatch-repair (MMR) systems correct all or almost all base-mismatch errors from DNA replication via excision-resynthesis pathways, and respond to many different DNA lesions. Consideration of DNA polymerase error rates and possible consequences of excess gratuitous excision of perfectly paired (homoduplex) DNA in vivo suggests that MMR needs to discriminate against homoduplex DNA by three to six orders of magnitude. However, numerous binding studies using MMR base-mispair-recognition proteins, bacterial MutS or eukaryotic MSH2.MSH6 (MutSalpha), have typically shown discrimination factors between mismatched and homoduplex DNA to be 5-30, depending on the binding conditions, the particular mismatches, and the DNA-sequence contexts. Thus, downstream post-binding steps must increase MMR discrimination without interfering with the versatility needed to recognize a large variety of base-mismatches and lesions. We use a complex but highly MMR-active model system, human nuclear extracts mixed with plasmid substrates containing specific mismatches and defined nicks 0.15 kbp away, to measure the earliest quantifiable committed step in mismatch correction, initiation of mismatch-provoked 3'-5' excision at the nicks. We compared these results to binding of purified MutSalpha to synthetic oligoduplexes containing the same mismatches in the same sequence contexts, under conditions very similar to those prevailing in the nuclear extracts. Discrimination against homoduplex DNA, only two-to five-fold in the binding studies, increased to 60- to 230-fold or more for excision initiation, depending on the particular mismatches. Remarkably, the mismatch-preference order for excision initiation was substantially altered from the order for hMutSalpha binding. This suggests that post-binding steps not only strongly discriminate against homoduplex DNA, but do so by mechanisms not tightly constrained by initial binding preferences. Pairs of homoduplexes (40, 50, and 70 bp) prepared from synthetic oligomers or cut out of plasmids showed virtually identical hMutSalpha binding affinities, suggesting that high hMutSalpha binding to homoduplex DNA is not the result of misincorporations or lesions introduced during chemical synthesis. Intrinsic affinities of MutS homologs for perfectly paired DNA may help these proteins efficiently position themselves to carry out subsequent mismatch-specific steps in MMR pathways.

Binding of MutS mismatch repair protein to DNA containing UV photoproducts, "mismatched" opposite Watson--Crick and novel nucleotides, in different DNA sequence contexts.

Mismatch-repair (MMR) systems suppress mutation via correction of DNA replication errors (base-mispairs) and responses to mutagenic DNA lesions. Selective binding of mismatched or damaged DNA by MutS-homolog proteins-bacterial MutS, eukaryotic MSH2.MSH6 (MutSalpha) and MSH2.MSH3-initiates mismatch-correction pathways and responses to lesions, and may cumulatively increase discrimination at downstream steps. MutS-homolog binding selectivity and the well-known but poorly understood effects of DNA-sequence contexts on recognition may thus be primary determinants of MMR specificity and efficiency. MMR processes that modulate UV mutagenesis might begin with selective binding by MutS homologs of "mismatched" T[CPD]T/AG and T[6--4]T/AG photoproducts, reported previously for hMutSalpha and described here for E. coli MutS protein. If MMR suppresses UV mutagenesis by acting directly on pre-mutagenic products of replicative bypass, mismatched photoproducts should be recognized in most DNA-sequence contexts. In three of four contexts tested here (three substantially different), T[CPD]T/AG was bound only slightly better by MutS than was T[CPD]T/AA or homoduplex DNA; only one of two contexts tested promoted selective binding of T[6--4]T/AG. Although the T:G pairs in T[CPD]T/AG and T/G both adopt wobble conformations, MutS bound T/G well in all contexts (K(1/2) 2.1--2.9 nM). Thus, MutS appears to select the two mismatches by different mechanisms. NMR analyses elsewhere suggest that in the (highly distorted) T[6--4]T/AG a forked H-bond between O2 of the 3' thymine and the ring 1-imino and exocyclic 2-amino guanine protons stabilizes a novel planar structure not possible in T[6--4]T/AA. Replacement of G by purines lacking one (inosine, 2-aminopurine) or both (nebularine) protons markedly reduced or eliminated selective MutS binding, as predicted. Previous studies and the work here, taken together, suggest that in only about half of DNA sequence contexts could MutS (and presumably MutSalpha) selectively bind mismatched UV photoproducts and directly suppress UV mutagenesis.

Testing excision models for responses of mismatch-repair systems to UV photoproducts in DNA.

Mismatch-repair (MMR) systems correct DNA replication errors and respond to a variety of DNA lesions. Previous observations that MMR antagonizes UV mutagenesis, and that the mismatch-recognition protein heterodimer MSH2*MSH6 (MutSalpha) selectively binds DNA containing "mismatched" photoproducts (T[CPD]T/AG, T[6-4]T/AG) but not "matched" photoproducts (T[CPD]T/AA, T[6-4]T/AA), suggested that mismatched photoproducts would provoke MMR excision similar to mismatched bases. Excision of incorrect nucleotides inserted opposite template photoproducts might then prevent UV-induced mutation. We tested T[CPD]T/AG DNA, in a sequence context in which it is bound substantially by hMutSalpha and in three other contexts, for stimulation of 3' MMR excision in mammalian nuclear extracts. T[CPD]T/AG was inactive in HeLa extracts, or in extracts deficient in the photoproduct-binding proteins DDB or XPC* hHR23B, arguing against interference from the nucleotide excision repair pathway. Prior incubation with hMutSalpha and MLH2.PMS2 (hMutLalpha) did not increase excision relative to homoduplex controls. T[6-4]T/AG also failed to provoke excision. T/G, C/A, and T/T substrates, even though bound by hMutSalpha no better than T[CPD]T/AG substrates, efficiently provoked excision. Even a substrate containing three T[CPD]T/AG photoproducts (in different contexts) did not significantly provoke excision. Thus, MMR may suppress UV mutagenesis by non-excisive mechanisms.

Transport stability of pesticides and PAHs sequestered in polyethylene passive sampling devices.

Research using low-density polyethylene (LDPE) passive samplers has steadily increased over the past two decades. However, such research efforts remain hampered because of strict guidelines, requiring that these samplers be quickly transported in airtight metal or glass containers or foil wrapped on ice. We investigate the transport stability of model pesticides and polycyclic aromatic hydrocarbons (PAHs) with varying physicochemical properties using polytetrafluoroethylene (PTFE) bags instead. Transport scenarios were simulated with transport times up to 14 days with temperatures ranging between -20 and 35 °C. Our findings show that concentrations of all model compounds examined were stable for all transport conditions tested, with mean recoveries ranging from 88 to 113 %. Furthermore, PTFE bags proved beneficial as reusable, lightweight, low-volume, low-cost alternatives to conventional containers. This documentation of stability will allow for more flexible transportation of LDPE passive samplers in an expanding range of research applications while maintaining experimental rigor.

Electron Capture Dissociation of Sodium-Adducted Peptides on a Modified Quadrupole/Time-of-Flight Mass Spectrometer.

Electron capture dissociation (ECD), which generally preserves the position of labile post-translational modifications, can be a powerful method for de novo sequencing of proteins and peptides. In this report, ECD product-ion mass spectra of singly and doubly sodiated, nonphosphorylated, and phosphorylated peptides are presented and compared with the ECD mass spectra of their protonated counterparts. ECD of doubly charged, singly sodiated peptides yielded essentially the same sequence information as was produced by the corresponding doubly protonated peptides. The presence of several sodium binding sites on the polypeptide backbone, however, resulted in more complicated spectra. This situation is aggravated by the zwitterionic equilibrium of the free acid peptide precursors. The product-ion spectra of doubly and triply charged peptides possessing two sodium ions were further complicated by the existence of isomers created by the differential distribution of sodium binding sites. Triply charged, phosphorylated precursors containing one sodium, wherein the sodium is attached exclusively to the PO4 group, were found to be as useful for sequence analysis as the fully protonated species. Although sodium adducts are generally minimized during sample preparation, it appears that they can nonetheless provide useful sequence information. Additionally, they enable straightforward identification of a peptide's charge state, even on low-resolution instruments. The experiments were carried out using a radio frequency-free electromagnetostatic cell retrofitted into the collision-induced dissociation (CID) section of a hybrid quadrupole/time-of-flight tandem mass spectrometer. Graphical Abstract ᅟ.

Modified ion source triple quadrupole mass spectrometer gas chromatograph for polycyclic aromatic hydrocarbon analyses.

We describe modified gas chromatography electron-impact/triple-quadrupole mass spectrometry (GC-EI/MS/MS) utilizing a newly developed hydrogen-injected self-cleaning ion source and modified 9mm extractor lens. This instrument, with optimized parameters, achieves quantitative separation of 62 polycyclic aromatic hydrocarbons (PAHs). Existing methods historically limited rigorous identification and quantification to a small subset, such as the 16 PAHs the US EPA has defined as priority pollutants. Without the critical source and extractor lens modifications, the off-the-shelf GC-EI/MS/MS system was unsuitable for complex PAH analysis. Separations were enhanced by increased gas flow, a complex GC temperature profile incorporating multiple isothermal periods, specific ramp rates, and a PAH-optimized column. Typical determinations with our refined GC-EI/MS/MS have a large linear range of 1-10,000pgµl(-1) and detection limits of <2pgµl(-1). Included in the 62 PAHs, multiple-reaction-monitoring (MRM) mode enabled GC-EI/MS/MS identification and quantitation of several constituents of the MW 302 PAH isomers. Using calibration standards, values determined were within 5% of true values over many months. Standard curve r(2) values were typically >0.998, exceptional for compounds which are archetypally difficult. With this method benzo[a]fluorene, benzo[b]fluorene, benzo[c]fluorene were fully separated as was benzo[b]fluoranthene, benzo[k]fluoranthene, and benzo[j]fluoranthene. Chrysene and triphenylene, were sufficiently separated to allow accurate quantitation. Mean limits of detection (LODs) across all PAHs were 1.02±0.84pgµl(-1) with indeno[1,2,3-c,d] pyrene having the lowest LOD at 0.26pgµl(-1) and only two analytes above 2.0pgµl(-1); acenaphthalene (2.33pgµl(-1)) and dibenzo[a,e]pyrene (6.44pgµl(-1)).

Dielectrophoresis and AC-induced assembly in binary colloidal suspensions.

Dielectrophoretic behaviors and assembly of a binary suspension in aqueous media are examined in the presence of nonuniform alternating current (AC) electric field. A peculiar low-frequency threshold and dielectrophoresis (DEP) crossover frequency determine the applicable frequency window for binary assembly under positive DEP, which can be effectively tuned by medium conductivity and particle size, suggesting that the dynamic double-layer effect is responsible for the interfacial polarization of micrometer to submicrometer-sized particles in aqueous suspensions. Strong effects of AC-field frequency, medium conductivity, and size ratio on binary assembly morphology have been observed. A frequency-medium conductivity phase diagram is obtained to illustrate the morphological transition of assembled colloidal aggregates from segregated, ordered assemblies to inverted segregation with the appearance of amorphous phases upon increasing frequency and/or medium conductivity, which is a direct consequence of the competition between DEP and hydrodynamic mobility. Significantly, our results demonstrate a rapid method to form hybrid nanostructured materials.

Biochemical evolution of DNA polymerase eta: properties of plant, human, and yeast proteins.

To assess how evolution might have biochemically shaped DNA polymerase eta (Poleta) in plants, we expressed in Escherichia coli proteins from Arabidopsis thaliana (At), humans (Hs), and the yeast Saccharomyces cerevisiae (Sc), purified them to near homogeneity, and compared their properties. Consistent with the multiple divergent amino acids within mostly conserved polymerase domains, the polymerases showed modest, appreciable, and marked differences, respectively, in salt and temperature optima for activity and thermostability. We compared abilities to extend synthetic primers past template cyclobutane thymine dimers (T[CPD]T) or undamaged T-T under physiological conditions (80-110 mM salt). Specific activities for "standing-start" extension of synthetic primers ending opposite the second template nucleotide 3' to T-T were roughly similar. During subsequent "running-start" insertions past T-T and the next 5' ( N + 1) nucleotide, AtPoleta and HsPoleta appeared more processive, but DNA sequence contexts strongly affected termination probabilities. Lesion-bypass studies employed four different templates containing T[CPD]Ts, and two containing pyrimidine (6-4')-pyrimidinone photoproducts ([6-4]s). AtPoleta made the three successive insertions [opposite the T[CPD]T and (N + 1) nucleotides] that define bypass nearly as well as HsPoleta and somewhat better than ScPoleta. Again, sequence context effects were profound. Interestingly, the level of insertion opposite the ( N - 1) nucleotide 3' to T[CPD]T by HsPoleta and especially AtPoleta, but not ScPoleta, was reduced (up to 4-fold) relative to the level of insertion opposite the ( N - 1) nucleotide 3' to T-T. Evolutionary conservation of efficient T[CPD]T bypass by HsPoleta and AtPoleta may reflect a high degree of exposure of human skin and plants to solar UV-B radiation. The depressed ( N - 1) insertion upstream of T[CPD]T (but not T-T) may reduce the extent of gratuitous error-prone insertion.

Rapid accumulation of mutations during seed-to-seed propagation of mismatch-repair-defective Arabidopsis.

During the many cell divisions that precede formation of plant gametes, their apical-meristem and floral antecedents are continually exposed to endogenous and environmental mutagenic threats. Although some deleterious recessive mutations may be eliminated during growth of haploid gametophytes and functionally haploid early embryos ("haplosufficiency quality-checking"), the multiplicity of plant genome-maintenance systems suggests aggressive quality control during prior diploid growth. To test in Arabidopsis a hypothesis that prior mismatch repair (MMR) is paramount in defense of plant genetic fidelity, we propagated in parallel 36 MMR-defective (Atmsh2-1) and 36 wild-type lines. The Atmsh2-1 lines rapidly accumulated a wide variety of mutations: fifth-generation (G5) plants showed abnormalities in morphology and development, fertility, germination efficiency, seed/silique development, and seed set. Only two Atmsh2-1, but all 36 wild-type lines, appeared normal at G5. Analyses of insertion/deletion mutation at six repeat-sequence (microsatellite) loci showed each Atmsh2-1 line to have evolved its own "fingerprint," the results of as many as 10 microsatellite mutations in a single line. Thus, MMR during diploid growth is essential for plant genomic integrity.