Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

Background: In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. Methodology/Principal Findings: We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkBssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. Conclusions/Significance: A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Sitedirected mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a ‘‘searching’ ’ mode and ‘‘repair’ ’ mode. Moreover, we are able to functionally separate these mode

Instantaneous Capture and Mineralization of Flue Gas Carbon Dioxide: Pilot Scale Study

Multiple CO2 capture and storage (CCS) processes are required to address anthropogenic CO2 problems. However, a method which can directly capture and mineralize CO2 at a point source, under actual field conditions, has advantages and could help offset the cost associated with the conventional CCS technologies. The mineral carbonation (MC), a process of converting CO2 into stable minerals (mineralization), has been studied extensively to store CO2. However, most of the MC studies have been largely investigated at laboratory scale. Objectives of this research were to develop a pilot scale AMC (accelerated mineral carbonation) process and test the effects of flue gas moisture content on carbonation of fly ash particles. A pilot scale AMC process consisting of a moisture reducing drum (MRD), a heater/humidifier, and a fluidized-bed reactor (FBR) was developed and tested by reacting flue gas with fly ash particles at one of the largest coal-fired power plants (2120 MW) in the USA. The experiments were conducted over a period of 2 hr at ~ 300 SCFM flow-rates, at a controlled pressure (115.1 kPa), and under different flue gas moisture contents (2-16%). The flue gas CO2 and SO2 concentrations were monitored before and during the experiments by an industrial grade gas analyzer. Fly ash samples were collected from the reactor sample port from 0-120 minutes and analyzed for total inorganic carbon (C), sulfur (S), and mercury (Hg). From C, S, and Hg concentrations, %calcium carbonate (CaCO3), %sulfate (SO42-), and %mercury carbonate (HgCO3) were calculated, respectively. Results suggested significant mineralization of flue gas CO2, SO2, and Hg within 10-15 minutes of reaction. Among different moisture conditions, ~16% showed highest conversion of flue gas CO2 and SO2 to %CaCO3 and %SO42- in fly ash samples. For example, an increase of almost 4% in CaCO3 content of fly ash was observed. Overall, the AMC process is cost-effective with minimum carbon footprint and can be retrofitted to coal fired power plants (existing and/or new) as a post-combustion unit to minimize flue gas CO2, SO2, and Hg emissions into the atmosphere. Used in conjunction with capture and geologic sequestration, the AMC process has the potential to reduce overall cost associated with CO2 separation/compression/transportation/pore space/brine water treatment. It could also help protect sensitive amines and carbon filters used in flue gas CO2 capture and separation process and extend their life

Synchronization of Developmental Processes and Defense Signaling by Growth Regulating Transcription Factors

Growth regulating factors (GRFs) are a conserved class of transcription factor in seed plants. GRFs are involved in various aspects of tissue differentiation and organ development. The implication of GRFs in biotic stress response has also been recently reported, suggesting a role of these transcription factors in coordinating the interaction between developmental processes and defense dynamics. However, the molecular mechanisms by which GRFs mediate the overlaps between defense signaling and developmental pathways are elusive. Here, we report large scale identification of putative target candidates of Arabidopsis GRF1 and GRF3 by comparing mRNA profiles of the grf1/grf2/grf3 triple mutant and those of the transgenic plants overexpressing miR396-resistant version of GRF1 or GRF3. We identified 1,098 and 600 genes as putative targets of GRF1 and GRF3, respectively. Functional classification of the potential target candidates revealed that GRF1 and GRF3 contribute to the regulation of various biological processes associated with defense response and disease resistance. GRF1 and GRF3 participate specifically in the regulation of defense-related transcription factors, cell-wall modifications, cytokinin biosynthesis and signaling, and secondary metabolites accumulation. GRF1 and GRF3 seem to fine-tune the crosstalk between miRNA signaling networks by regulating the expression of several miRNA target genes. In addition, our data suggest that GRF1 and GRF3 may function as negative regulators of gene expression through their association with other transcription factors. Collectively, our data provide new insights into how GRF1 and GRF3 might coordinate the interactions between defense signaling and plant growth and developmental pathways

Cooperative Regulatory Functions of miR858 and MYB83 during Cyst Nematode Parasitism

MicroRNAs (miRNAs) recently have been established as key regulators of transcriptome reprogramming that define cell function and identity. Nevertheless, the molecular functions of the greatest number of miRNA genes remain to be determined. Here, we report cooperative regulatory functions of miR858 and its MYB83 transcription factor target gene in transcriptome reprogramming during Heterodera cyst nematode parasitism of Arabidopsis (Arabidopsis thaliana). Gene expression analyses and promoter-GUS fusion assays documented a role of miR858 in posttranscriptional regulation of MYB83 in the Heterodera schachtii-induced feeding sites, the syncytia. Constitutive overexpression of miR858 interfered with H. schachtii parasitism of Arabidopsis, leading to reduced susceptibility, while reduced miR858 abundance enhanced plant susceptibility. Similarly, MYB83 expression increases were conducive to nematode infection because overexpression of a noncleavable coding sequence of MYB83 significantly increased plant susceptibility, whereas a myb83 mutation rendered the plants less susceptible. In addition, RNA-seq analysis revealed that genes involved in hormone signaling pathways, defense response, glucosinolate biosynthesis, cell wall modification, sugar transport, and transcriptional control are the key etiological factors by which MYB83 facilitates nematode parasitism of Arabidopsis. Furthermore, we discovered that miR858-mediated silencing of MYB83 is tightly regulated through a feedback loop that might contribute to fine-tuning the expression of more than a thousand of MYB83-regulated genes in the H. schachtii-induced syncytium. Together, our results suggest a role of the miR858-MYB83 regulatory system in finely balancing gene expression patterns during H. schachtii parasitism of Arabidopsis to ensure optimal cellular function

Arabidopsis miR827 mediates post‐transcriptional gene silencing of its ubiquitin E3 ligase target gene in the syncytium of the cyst nematode Heterodera schachtii to enhance susceptibility

MicroRNAs (miRNAs) are a major class of small non-coding RNAs with emerging functions in biotic and abiotic interactions. Here, we report on a new functional role of Arabidopsis miR827 and its NITROGEN LIMITATION ADAPTATION (NLA) target gene in mediating plant susceptibility to the beet cyst nematode Heterodera schachtii. Cyst nematodes are sedentary endoparasites that induce the formation of multinucleated feeding structures termed syncytia in the roots of host plants. Using promoter:GUS fusion assays we established that miR827 was activated in the initial feeding cells and this activation was maintained in the syncytium during all sedentary stages of nematode development. Meanwhile, the NLA target gene, which encodes an ubiquitin E3 ligase enzyme, was post-transcriptionally silenced in the syncytium, to permanently suppress its activity during all nematode parasitic stages. Overexpression of miR827 in Arabidopsis resulted in hyper-susceptibility to H. schachtii. In contrast, inactivation of miR827 activity through target mimicry or by overexpression a miR827-resistant cDNA of NLA produced the opposite phenotype of reduced plant susceptibility to H. schachtii. Gene expression analysis of several pathogenesis-related genes together with Agrobacterium–mediated transient expression in Nicotiana benthamiana provided strong evidence that miR827-mediated downregulation of NLA to suppress basal defense pathways. In addition, using yeast two-hybrid screens we identified several candidates of NLA-interacting proteins that are involved in a wide range of biological processes and molecular functions, including three pathogenesis-related proteins. Taken together, we conclude that nematode-activated miR827 in the syncytium is necessary to suppress immune responses in order to establish infection and cause disease

Investigating whether adverse prenatal and perinatal events are associated with non-clinical psychotic symptoms at age 12 years in the ALSPAC birth cohort

Background. Non-clinical psychosis-like symptoms (PLIKS) occur in about 15% of the population. It is not clear whether adverse events during early development alter the risk of developing PLIKS. We aimed to examine whether maternal infection, diabetes or pre-eclampsia during pregnancy, gestational age, perinatal cardiopulmonary resuscitation or 5-min Apgar score were associated with development of psychotic symptoms during early adolescence. Method. A longitudinal study of 6356 12-year-old adolescents who completed a semi-structured interview for psychotic symptoms in the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort. Prenatal and perinatal data were obtained from obstetric records and maternal questionnaires completed during pregnancy. Results. The presence of definite psychotic symptoms was associated with maternal infection during pregnancy [adjusted odds ratio (OR) 1.44, 95% confidence interval (CI) 1.11–1.86, p=0.006], maternal diabetes (adjusted OR 3.43, 95% CI 1.14–10.36, p=0.029), need for resuscitation (adjusted OR 1.50, 95% CI 0.97–2.31, p=0.065) and 5-min Apgar score (adjusted OR per unit decrease 1.30, 95% CI 1.12–1.50, p<0.001). None of these associations were mediated by childhood IQ score. Most associations persisted, but were less strong, when including suspected symptoms as part of the outcome. There was no association between PLIKS and gestational age or pre-eclampsia. Conclusions. Adverse events during early development may lead to an increased risk of developing PLIKS. Although the status of PLIKS in relation to clinical disorders such as schizophrenia is not clear, the similarity between these results and findings reported for schizophrenia indicates that future studies of PLIKS may help us to understand how psychotic experiences and clinical disorders develop throughout the life-course

Studies of a Lacustrine-Volcanic Mars Analog Field Site with Mars-2020-like Instruments

On the upcoming Mars‐2020 rover two remote sensing instruments, Mastcam‐Z and SuperCam, and two microscopic proximity science instruments, SHERLOC and PIXL, will collect compositional (mineralogy, chemistry, and organics) data essential for paleoenvironmental reconstruction. The synergies between and limitations of these instruments were evaluated via study of a Mars analog field site in the Mojave Desert, using instruments approximating the data that will be returned by Mars‐2020. A ground truth dataset was generated for comparison to validate the results. The site consists of a succession of clay‐rich mudstones of lacustrine origin, interbedded tuffs, a carbonate‐silica travertine deposit, and gypsiferous mudstone strata. The major geological units were mapped successfully using simulated Mars‐2020 data. Simulated Mastcam‐Z data identified unit boundaries and Fe‐bearing weathering products. Simulated SuperCam passive shortwave infrared and green Raman data were essential in identifying major mineralogical composition and changes in lacustrine facies at distance; this was possible even with spectrally downsampled passive IR data. LIBS and simulated PIXL data discriminated and mapped major element chemistry. Simulated PIXL revealed mm‐scale zones enriched in zirconium, of interest for age dating. SHERLOC‐like data mapped sulfate and carbonate at sub‐mm scale; silicates were identified with increased laser pulses/spot or by averaging of hundreds of spectra. Fluorescence scans detected and mapped varied classes of organics in all samples, characterized further with follow‐on spatially targeted deep‐UV Raman spectra. Development of dedicated organics spectral libraries is needed to aid interpretation. Given these observations, the important units in the outcrop would be sampled and cached for sample return

Vitamin D Status Relative to Diet, Lifestyle, Injury, and Illness in College Athletes

Vitamin D deficiency is endemic in the general population; however, there is much to be learned about the vitamin D status of athletes. Purpose: The purposes of this study were to assess the prevalence of vitamin D insufficiency in collegiate athletes and to determine whether 25(OH)D concentrations are related to vitamin D intake, sun exposure, body composition, and risk for illness or athletic injury. Methods: 25(OH) vitamin D concentrations were measured in 41 athletes (18 men/23 women, 12 indoor/29 outdoor athletes) throughout the academic year. Dietary intake and lifestyle habits were assessed via questionnaire, bone density was measured by dual energy x-ray absorptiometry, and injury and illness were documented as part of routine care. Results: The 25(OH)D concentrations changed across time (P = 0.001) and averaged 49.0 T 16.6, 30.5 T 9.4, and 41.9 T 14.6 ngImLj1 (mean T SD) in the fall, winter, and spring, respectively, and were higher in outdoor versus indoor athletes in the fall (P G 0.05). Using 40 ngImLj1 as the cutoff for optimal status, 75.6%, 15.2%, and 36.0% of athletes had optimal status in the fall, winter, and spring, respectively. 25(OH)D concentrations were significantly (P G 0.05) correlated with multivitamin intake in the winter (r = 0.39) and tanning bed use in the spring (r = 0.48); however, status was otherwise not related to intake, lifestyle factors, or body composition. 25(OH)D concentrations in the spring (r = j0.40, P = 0.048) was correlated with frequency of illness. Conclusions: Our results suggest that collegiate athletes can maintain sufficient status during the fall and spring but would benefit from supplementation during the winter to prevent seasonal decreases in 25(OH)D concentrations. Results further suggest that insufficient vitamin D status may increase risk for frequent illness. Future research is needed to identify whether vitamin D status influences injury risk during athletic training or competition

Analyzing Adherence to Prenatal Supplement: Does Pill Count Measure Up?

Objective. To determine if adherence as measured by pill count would show a significant association with serum-based measures of adherence. Methods. Data were obtained from a prenatal vitamin D supplementation trial where subjects were stratified by race and randomized into three dosing groups: 400 (control), 2000, or 4000 IU vitamin D3/day. One measurement of adherence was obtained via pill counts remaining compared to a novel definition for adherence using serum 25-hydroxy-vitamin D (25-OH-D) levels (absolute change in 25(OH)D over the study period and the subject's steady-state variation in their 25(OH)D levels). A multivariate logistic regression model examined whether mean percent adherence by pill count was significantly associated with the adherence measure by serum metabolite levels. Results. Subjects' mean percentage of adherence by pill count was not a significant predictor of adherence by serum metabolite levels. This finding was robust across a series of sensitivity analyses. Conclusions. Based on our novel definition of adherence, pill count was not a reliable predictor of adherence to protocol, and calls into question how adherence is measured in clinical research. Our findings have implications regarding the determination of efficacy of medications under study and offer an alternative approach to measuring adherence of long half-life supplements/medications