The LTAR cropland common experiment in the Lower Mississippi River Basin.

The Lower Mississippi River Basin-Long-Term Agroecosystem Research Site (LMRB-LTAR) encompasses six states from Missouri to the Gulf of Mexico and is coordinated by the USDA-ARS National Sedimentation Laboratory, Oxford, MS. The overarching goal of LTAR is to assess regionally diverse and geographically scalable farming practices for enhanced sustainability of agroecosystem goods and services under changing environment and resource-use conditions. The LMRB-LTAR overall goal is to assess sustainable row crop agricultural production systems that integrate regional environmental and socioeconomic needs. Primary row crops in the region include soybeans, corn, cotton, rice, and sugarcane with crop rotations influenced by commodity crop price and other factors. The field-scale common experiment (CE) includes four row crop farms (26-101 ha) established in 2021 and 2023. Three fields are managed with alternative practices, including reduced tillage, cover crops, and automated prescription irrigation, and three fields are managed with prevailing farming practices, consisting of conventional tillage, no cover crop, and nonprescription irrigation. Treatment effects on crop productivity, soil quality, water use efficiency, water quality, and carbon storage are assessed. Research from the LMRB CE will deliver outcomes linked to overarching LTAR network goals, including innovative agricultural systems, strengthened partnerships, data management technologies, and precision environmental tools.

Reaction time as an outcome measure during online fear conditioning: Effects of number of trials, age, and levels of processing.

Recent studies have shown that fear conditioning experiments can be successfully conducted online. However, there is limited evidence that measures other than subjective ratings of threat expectancy can be collected, which means that online research may not be able to adequately replace laboratory experiments. In the current study, we conducted an online fear conditioning experiment consisting of habituation, acquisition, extinction and 48 h delayed extinction recall using ratings of threat expectancy and conditional stimulus pleasantness, and probe reaction time as outcome measures. The conditional stimuli were categories of words and a levels of processing manipulation explored whether words that were processed at a deeper level during extinction evoked smaller differential threat responses during extinction recall. Although the levels of processing manipulation did not produce a significant outcome, we found that extinction recall was successfully operationalised in our study. Reaction time indicated differential responding during both acquisition and extinction recall, and age of participants was correlated in one of two experiments with differential threat expectancy and reaction time, such that older participants showed better safety learning. The outcomes of this experiment provide multiple novel tools for researchers to explore fear conditioning, especially in an online environment.

Evaluating a large language model's ability to solve programming exercises from an introductory bioinformatics course.

Computer programming is a fundamental tool for life scientists, allowing them to carry out essential research tasks. However, despite various educational efforts, learning to write code can be a challenging endeavor for students and researchers in life-sciences disciplines. Recent advances in artificial intelligence have made it possible to translate human-language prompts to functional code, raising questions about whether these technologies can aid (or replace) life scientists' efforts to write code. Using 184 programming exercises from an introductory-bioinformatics course, we evaluated the extent to which one such tool-OpenAI's ChatGPT-could successfully complete programming tasks. ChatGPT solved 139 (75.5%) of the exercises on its first attempt. For the remaining exercises, we provided natural-language feedback to the model, prompting it to try different approaches. Within 7 or fewer attempts, ChatGPT solved 179 (97.3%) of the exercises. These findings have implications for life-sciences education and research. Instructors may need to adapt their pedagogical approaches and assessment techniques to account for these new capabilities that are available to the general public. For some programming tasks, researchers may be able to work in collaboration with machine-learning models to produce functional code.

Arthroscopic debridement for management of massive, irreparable rotator cuff tears: a systematic review of outcomes.

Background: Surgical management of massive irreparable rotator cuff tears remains controversial. Arthroscopic debridement (AD) has shown promising results especially in the population older than 65 years; however, there is no consensus on the benefits of various AD procedures. The aim of this systematic review was to evaluate the functional midterm to long-term outcomes in patients treated with AD in combination with subacromial decompression, biceps tenotomy, tuberoplasty, or bursectomy, without repair of the rotator cuff tear. Methods: A comprehensive search was performed in PubMed, Cumulative Index to Nursing and Allied Health Literature (CINAHL), and Cochrane databases for studies reporting clinical outcomes of AD of massive rotator cuff tears. Quality was determined using the Methodological Index for Nonrandomized Studies (MINORS) criteria by two independent reviewers. Pooled frequency-weighted means and standard deviations were calculated for patient-reported outcomes. Results: Sixteen articles containing 643 patients and 662 shoulders met the eligibility criteria. The mean age at the time of surgery was 65.9 ± 4.4 years with a mean follow-up period of 46.5 ± 27.3 months. There was notable clinically significant improvement across all patient-reported outcome scores postoperatively: Constant 70.4 ± 8.9 (P value = .06), University of California, Los Angeles ultrasonography 26.7 ± 5.2 (P value = .001), American Shoulder and Elbow Surgeons score 71.7 ± 2.1 (P value = .12), Disabilities of the Arm, Shoulder, and Hand score 35.3, and visual analog score 1.7 ± 0.9. Forty-nine patients (7%) required reoperation, which most commonly was a reverse total shoulder arthroplasty for the development of rotator cuff arthropathy. Conclusion: Arthroscopic debridement with a combination of subacromial decompression, tuberoplasty, subacromial bursectomy, and biceps tenotomy, for treatment of massive irreparable rotator cuff tears, produces good functional outcomes and improvement in pain at mid to long term follow up for the low-demand population greater than 65 years of age looking for pain relief over substantial increase in function.

Correction: Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation.

[This corrects the article DOI: 10.1371/journal.pone.0198154.].

Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation.

A critical issue in transgene delivery studies is immune reactivity to the transgene- encoded protein and its impact on sustained gene expression. Here, we test the hypothesis that immunomodulation by rapamycin can decrease immune reactivity after intrathecal AAV9 delivery of a transgene (GFP) in non-human primates, resulting in sustained GFP expression in the CNS. We show that rapamycin treatment clearly reduced the overall immunogenicity of the AAV9/GFP vector by lowering GFP- and AAV9-specific antibody responses, and decreasing T cell responses including cytokine and cytolytic effector responses. Spinal cord GFP protein expression was sustained for twelve weeks, with no toxicity. Immune correlates of robust transgene expression include negligible GFP-specific CD4 and CD8 T cell responses, absence of GFP-specific IFN-γ producing T cells, and absence of GFP-specific cytotoxic T cells, which support the hypothesis that decreased T cell reactivity results in sustained transgene expression. These data strongly support the use of modest doses of rapamycin to modulate immune responses for intrathecal gene therapies, and potentially a much wider range of viral vector-based therapeutics.

Analysis of septic biomarker patterns: prognostic value in predicting septic state.

Patients with infection, sepsis, severe sepsis, or septic shock were compared to each other and to healthy controls with regard to serum levels of biomarkers and clinical symptoms. Of the 15 biomarkers assayed, 9 were detectable in patients, and 4, in controls. Both proinflammatory and anti-inflammatory cytokines were detected in the patients. No single biomarker could differentiate the 3 septic levels of severity from each other; however, interleukin (IL) 1 receptor antagonist (IL-1ra) had the best sensitivity and specificity for differentiating sepsis and severe sepsis from septic shock. IL-6 was the only cytokine able to differentiate infected patients without signs of sepsis from those with sepsis. Although IL-1ra, IL-6, IL-8, and monocyte chemoattractant protein 1 could differentiate infection, sepsis, and severe sepsis from septic shock, the biomarkers could not differentiate sepsis from severe sepsis. The top scoring pair algorithm with clinical and biomarker analyses was able to correctly diagnose those with sepsis who will progress to a more severe state.

Transcriptional dysregulation of inflammatory/immune pathways after active vaccination against Huntington's disease.

Immunotherapy, both active and passive, is increasingly recognized as a powerful approach to a wide range of diseases, including Alzheimer's and Parkinson's. Huntington's disease (HD), an autosomal dominant disorder triggered by misfolding of huntingtin (HTT) protein with an expanded polyglutamine tract, could also benefit from this approach. Individuals can be identified genetically at the earliest stages of disease, and there may be particular benefits to a therapy that can target peripheral tissues in addition to brain. In this active vaccination study, we first examined safety and immunogenicity for a broad series of peptide, protein and DNA plasmid immunization protocols, using fragment (R6/1), and knock-in (zQ175) models. No safety issues were found. The strongest and most uniform immune response was to a combination of three non-overlapping HTT Exon1 coded peptides, conjugated to KLH, delivered with alum adjuvant. An N586-82Q plasmid, delivered via gene gun, also showed ELISA responses, mainly in the zQ175 strain, but with more variability, and less robust responses in HD compared with wild-type controls. Transcriptome profiling of spleens from the triple peptide-immunized cohort showed substantial HD-specific differences including differential activation of genes associated with innate immune responses, absence of negative feedback control of gene expression by regulators, a temporal dysregulation of innate immune responses and transcriptional repression of genes associated with memory T cell responses. These studies highlight critical issues for immunotherapy and HD disease management in general.

A Quantitative Measure of Field Illumination.

In this paper, we describe a statistically based algorithm to quantify the uniformity of illumination in an optical light microscopy imaging system that outputs a single quality factor (QF) score. The importance of homogeneous field illumination in quantitative light microscopy is well understood and often checked. However, there is currently no standard automatic quantitative measure of the uniformity of the field illumination. Images from 89 different laser-scanning confocal microscopes (LSCMs), which were collected as part of an international study on microscope quality assessment, were used as a "training" set to build the algorithm. To validate the algorithm and verify its robustness, images from 33 additional microscopes, including LSCM and wide-field (WF) microscopes, were used. The statistical paradigm used for developing the quality scoring scale was a regression approach to supervised learning. Three intensity profiles across each image-2 corner-to-corner diagonals and a center horizontal-were used to generate pixel-intensity data. All of the lines passed through the center of the image. The intensity profile data then were converted into a single-field illumination QF score in the range of 0-100, with 0 having extreme variation, and therefore, essentially unusable, and 100 having no deviation, i.e., straight lines with a constant uniform intensity. Empirically, a QF ≥ 83 was determined to be the minimum acceptable value based on manufacturer acceptance tests and reasonably achievable values. This new QF is an invaluable metric to ascertain objectively and easily the uniformity of illumination quality, provide a traceable reference for monitoring field uniformity over time, and make a direct comparison among different microscopes. The QF can also be used as an indicator of system failure and the need for alignment or service of the instrument.

Angiogenesis QTL on Mouse Chromosome 8 Colocalizes with Differential ß-Defensin Expression.

Identification of genetic factors that modify complex traits is often complicated by gene-environment interactions that contribute to the observed phenotype. In model systems, the phenotypic outcomes quantified are typically traits that maximize observed variance, which in turn, should maximize the detection of quantitative trait loci (QTL) in subsequent mapping studies. However, when the observed trait is dependent on multiple interacting factors, it can complicate genetic analysis, reducing the likelihood that the modifying mutation will ultimately be found. Alternatively, by focusing on intermediate phenotypes of a larger condition, we can reduce a model's complexity, which will, in turn, limit the number of QTL that contribute to variance. We used a novel method to follow angiogenesis in mice that reduces environmental variance by measuring endothelial cell growth from culture of isolated skin biopsies that varies depending on the genetic source of the tissue. This method, in combination with a backcross breeding strategy, is intended to reduce genetic complexity and limit the phenotypic effects to fewer modifier loci. We determined that our approach was an efficient means to generate recombinant progeny and used this cohort to map a novel s.c. angiogenesis QTL to proximal mouse chromosome (Chr.) 8 with suggestive QTL on Chr. 2 and 7. Global mRNA expression analysis of samples from parental reference strains revealed ß-defensins as potential candidate genes for future study.

Genome-scale analyses of Escherichia coli and Salmonella enterica AraC reveal noncanonical targets and an expanded core regulon.

Escherichia coli AraC is a well-described transcription activator of genes involved in arabinose metabolism. Using complementary genomic approaches, chromatin immunoprecipitation (ChIP)-chip, and transcription profiling, we identify direct regulatory targets of AraC, including five novel target genes: ytfQ, ydeN, ydeM, ygeA, and polB. Strikingly, only ytfQ has an established connection to arabinose metabolism, suggesting that AraC has a broader function than previously described. We demonstrate arabinose-dependent repression of ydeNM by AraC, in contrast to the well-described arabinose-dependent activation of other target genes. We also demonstrate unexpected read-through of transcription at the Rho-independent terminators downstream of araD and araE, leading to significant increases in the expression of polB and ygeA, respectively. AraC is highly conserved in the related species Salmonella enterica. We use ChIP sequencing (ChIP-seq) and RNA sequencing (RNA-seq) to map the AraC regulon in S. enterica. A comparison of the E. coli and S. enterica AraC regulons, coupled with a bioinformatic analysis of other related species, reveals a conserved regulatory network across the family Enterobacteriaceae comprised of 10 genes associated with arabinose transport and metabolism.

The maternal autoimmune environment affects the social behavior of offspring.

Autism spectrum disorders (ASD) are neurodevelopmental disorders with unknown etiology. BTBR-T(+)tf/J (BTBR) mice, a mouse strain with behaviors that resemble autism and with elevated levels of anti-brain antibodies (Abs), have enhanced activation of peripheral B cells and CD4(+) T cells and an expanded percentage of CD4(+) T cells expressing Vß6 chains. The CD4(+)CD25(+)Vß6(+) and Vß6-splenic cells of BTBR mice have elevated levels of IL-4, IFN-γ and IL-17, but there appears to be no preferential CD4(+) T subset skewing/polarization. The high level of IgG production by BTBR B cells was dependent on T cells from BTBR mice. The CD4(+) T cells of BTBR mice, especially those expressing Vß6 become spontaneously activated and expanded in an autoimmune-like manner, which occurred in both BTBR and B6 hosts that received an equal number of BTBR and B6 bone marrow cells. BTBR mice also have an elevated percentage of peripheral blood neutrophils, which may represent their elevated inflammatory state. B6 offspring derived from B6 dams that were gestationally injected with purified IgG from sera of BTBR mice, but not IgG of B6 mice, developed significantly impaired social behavior. Additionally, B6 offspring that developed in BTBR dams had impaired social behavior, while BTBR offspring that developed in B6 dams had improved social behavior. All of the immunological and behavioral parameters of BTBR mice were compared with those of B6 mice, which have relatively normal behaviors. The results indicate maternal Abs and possibly other maternal influences affect the social behavior of offspring.

Nutrient removal using biosorption activated media: preliminary biogeochemical assessment of an innovative stormwater infiltration basin.

Soil beneath a stormwater infiltration basin receiving runoff from a 23 ha predominantly residential watershed in north-central Florida, USA, was amended using biosorption activated media (BAM) to study the effectiveness of this technology in reducing inputs of nitrogen and phosphorus to groundwater. The functionalized soil amendment BAM consists of a 1.0:1.9:4.1 mixture (by volume) of tire crumb (to increase sorption capacity), silt and clay (to increase soil moisture retention), and sand (to promote sufficient infiltration), which was applied to develop an innovative stormwater infiltration basin utilizing nutrient reduction and flood control sub-basins. Comparison of nitrate/chloride (NO(3)(-)/Cl(-)) ratios for the shallow groundwater indicates that prior to using BAM, NO(3)(-) concentrations were substantially influenced by nitrification or variations in NO(3)(-) input. In contrast, for the new basin utilizing BAM, NO(3)(-)/Cl(-) ratios indicate minor nitrification and NO(3)(-) losses with the exception of one summer sample that indicated a 45% loss. Biogeochemical indicators (denitrifier activity derived from real-time polymerase chain reaction and variations in major ions, nutrients, dissolved and soil gases, and stable isotopes) suggest that NO(3)(-) losses are primarily attributable to denitrification, whereas dissimilatory nitrate reduction to ammonium is a minor process. Denitrification was likely occurring intermittently in anoxic microsites in the unsaturated zone, which was enhanced by the increased soil moisture within the BAM layer and resultant reductions in surface/subsurface oxygen exchange that produced conditions conducive to increased denitrifier activity. Concentrations of total dissolved phosphorus and orthophosphate (PO(4)(3-)) were reduced by more than 70% in unsaturated zone soil water, with the largest decreases in the BAM layer where sorption was the most likely mechanism for removal. Post-BAM PO(4)(3-)/Cl(-) ratios for shallow groundwater indicate predominantly minor increases and decreases in PO(4)(3-) with the exception of one summer sample that indicated a 50% loss. Differences in nutrient variations between the unsaturated zone and shallow groundwater may be the result of the intensity and duration of nutrient removal processes and mixing ratios with water that had undergone little biogeochemical transformation. Observed nitrogen and phosphorus losses demonstrate the potential, as well as the future research needs to improve performance, of the innovative stormwater infiltration basin using BAM for providing passive, economical, stormwater nutrient-treatment technology to support green infrastructure.

Cyclic biogeochemical processes and nitrogen fate beneath a subtropical stormwater infiltration basin.

A stormwater infiltration basin in north-central Florida, USA, was monitored from 2007 through 2008 to identify subsurface biogeochemical processes, with emphasis on N cycling, under the highly variable hydrologic conditions common in humid, subtropical climates. Cyclic variations in biogeochemical processes generally coincided with wet and dry hydrologic conditions. Oxidizing conditions in the subsurface persisted for about one month or less at the beginning of wet periods with dissolved O(2) and NO(3)(-) showing similar temporal patterns. Reducing conditions in the subsurface evolved during prolonged flooding of the basin. At about the same time O(2) and NO(3)(-) reduction concluded, Mn, Fe and SO(4)(2-) reduction began, with the onset of methanogenesis one month later. Reducing conditions persisted up to six months, continuing into subsequent dry periods until the next major oxidizing infiltration event. Evidence of denitrification in shallow groundwater at the site is supported by median NO(3)(-)-N less than 0.016 mg L(-1), excess N(2) up to 3 mg L(-1) progressively enriched in δ(15)N during prolonged basin flooding, and isotopically heavy δ(15)N and δ(18)O of NO(3)(-) (up to 25‰ and 15‰, respectively). Isotopic enrichment of newly infiltrated stormwater suggests denitrification was partially completed within two days. Soil and water chemistry data suggest that a biogeochemically active zone exists in the upper 1.4m of soil, where organic carbon was the likely electron donor supplied by organic matter in soil solids or dissolved in infiltrating stormwater. The cyclic nature of reducing conditions effectively controlled the N cycle, switching N fate beneath the basin from NO(3)(-) leaching to reduction in the shallow saturated zone. Results can inform design of functionalized soil amendments that could replace the native soil in a stormwater infiltration basin and mitigate potential NO(3)(-) leaching to groundwater by replicating the biogeochemical conditions under the observed basin.

Dynamics of molecular responses to coxsackievirus B4 infection differentiate between resolution and progression of acute pancreatitis.

A coxsackievirus B4 induces acute pancreatitis with different outcomes. The study utilized a systems biology approach to identify molecular immune responses that differentiate between disease resolution and disease progression. The data establish a temporal pattern of host responses that differentiate the resolution of acute pancreatitis from the progression to chronic pancreatitis. A group of twenty-five genes exhibited characteristic expression profiles that were observed during the development of chronic pancreatitis but not during the resolution of disease. We postulate that the temporal dynamics of the twenty-five genes influence the development of pathogenic immune responses associated with chronic pancreatitis. Furthermore, a subset of eleven genes exhibited increased expression as viral titers waned. Of the eleven gene products, five are secreted molecules, TNF-α, IFN-γ, CXCL10, IL-10, and IL-22b, and represent novel potential therapeutic targets since they can be readily modulated with antibodies against the specific cytokine/chemokine or with antibodies against the corresponding receptors.

Soil property control of biogeochemical processes beneath two subtropical stormwater infiltration basins.

Substantially different biogeochemical processes affecting nitrogen fate and transport were observed beneath two stormwater infiltration basins in north-central Florida. Differences are related to soil textural properties that deeply link hydroclimatic conditions with soil moisture variations in a humid, subtropical climate. During 2008, shallow groundwater beneath the basin with predominantly clayey soils (median, 41% silt+clay) exhibited decreases in dissolved oxygen from 3.8 to 0.1 mg L and decreases in nitrate nitrogen (NO-N) from 2.7 mg L to <0.016 mg L, followed by manganese and iron reduction, sulfate reduction, and methanogenesis. In contrast, beneath the basin with predominantly sandy soils (median, 2% silt+clay), aerobic conditions persisted from 2007 through 2009 (dissolved oxygen, 5.0-7.8 mg L), resulting in NO-N of 1.3 to 3.3 mg L in shallow groundwater. Enrichment of δN and δO of NO combined with water chemistry data indicates denitrification beneath the clayey basin and relatively conservative NO transport beneath the sandy basin. Soil-extractable NO-N was significantly lower and the copper-containing nitrite reductase gene density was significantly higher beneath the clayey basin. Differences in moisture retention capacity between fine- and coarse-textured soils resulted in median volumetric gas-phase contents of 0.04 beneath the clayey basin and 0.19 beneath the sandy basin, inhibiting surface/subsurface oxygen exchange beneath the clayey basin. Results can inform development of soil amendments to maintain elevated moisture content in shallow soils of stormwater infiltration basins, which can be incorporated in improved best management practices to mitigate NO impacts.

High-throughput sequencing of Astrammina rara: sampling the giant genome of a giant foraminiferan protist.

BACKGROUND: Foraminiferan protists, which are significant players in most marine ecosystems, are also genetic innovators, harboring unique modifications to proteins that make up the basic eukaryotic cell machinery. Despite their ecological and evolutionary importance, foraminiferan genomes are poorly understood due to the extreme sequence divergence of many genes and the difficulty of obtaining pure samples: exogenous DNA from ingested food or ecto/endo symbionts often vastly exceed the amount of "native" DNA, and foraminiferans cannot be cultured axenically. Few foraminiferal genes have been sequenced from genomic material, although partial sequences of coding regions have been determined by EST studies and mass spectroscopy. The lack of genomic data has impeded evolutionary and cell-biology studies and has also hindered our ability to test ecological hypotheses using genetic tools. RESULTS: 454 sequence analysis was performed on a library derived from whole genome amplification of microdissected nuclei of the Antarctic foraminiferan Astrammina rara. Xenogenomic sequence, which was shown not to be of eukaryotic origin, represented only 12% of the sample. The first foraminiferal examples of important classes of genes, such as tRNA genes, are reported, and we present evidence that sequences of mitochondrial origin have been translocated to the nucleus. The recovery of a 3' UTR and downstream sequence from an actin gene suggests that foraminiferal mRNA processing may have some unusual features. Finally, the presence of a co-purified bacterial genome in the library also permitted the first calculation of the size of a foraminiferal genome by molecular methods, and statistical analysis of sequence from different genomic sources indicates that low-complexity tracts of the genome may be endoreplicated in some stages of the foraminiferal life cycle. CONCLUSIONS: These data provide the first window into genomic organization and genetic control in these organisms, and also complement and expands upon information about foraminiferal genes based on EST projects. The genomic data obtained are informative for environmental and cell-biological studies, and will also be useful for efforts to understand relationships between foraminiferans and other protists.

IL-10 is pathogenic during the development of coxsackievirus B4-induced chronic pancreatitis.

Using a mouse model of coxsackievirus B4 (CVB4-V)-induced chronic pancreatitis, we investigated whether cytokines are involved in the progression of acute disease to chronic inflammatory disease. We show that IL-10 contributed to the development of chronic pancreatitis since acute disease resolved when IL-10 was absent or when IL-10 signaling was disrupted. We explored the underlying mechanisms by which IL-10 affected disease progression, using a novel approach to assess immunological events occurring in situ. Multiple markers that define functional innate immune responses and functional T cell responses were monitored over the course of CVB4-V infection of wild-type and IL-10 knockout mice, using a multiplex transcriptional profiling approach. We show that high levels of IL-10 early during infection were associated with delayed innate and T cell responses. Furthermore, high IL-10 production correlated with altered kinetics of T regulatory responses indicating a disruption in the balance between effector and regulatory T cell responses.

Gene promoter methylation assayed in exhaled breath, with differences in smokers and lung cancer patients.

BACKGROUND: There is a need for new, noninvasive risk assessment tools for use in lung cancer population screening and prevention programs. METHODS: To investigate the technical feasibility of determining DNA methylation in exhaled breath condensate, we applied our previously-developed method for tag-adapted bisulfite genomic DNA sequencing (tBGS) for mapping of DNA methylation, and adapted it to exhaled breath condensate (EBC) from lung cancer cases and non-cancer controls. Promoter methylation patterns were analyzed in DAPK, RASSF1A and PAX5beta promoters in EBC samples from 54 individuals, comprised of 37 controls [current- (n = 19), former- (n = 10), and never-smokers (n = 8)] and 17 lung cancer cases [current- (n = 5), former- (n = 11), and never-smokers (n = 1)]. RESULTS: We found: (1) Wide inter-individual variability in methylation density and spatial distribution for DAPK, PAX5beta and RASSF1A. (2) Methylation patterns from paired exhaled breath condensate and mouth rinse specimens were completely divergent. (3) For smoking status, the methylation density of RASSF1A was statistically different (p = 0.0285); pair-wise comparisons showed that the former smokers had higher methylation density versus never smokers and current smokers (p = 0.019 and p = 0.031). For DAPK and PAX5beta, there was no such significant smoking-related difference. Underlying lung disease did not impact on methylation density for this geneset. (4) In case-control comparisons, CpG at -63 of DAPK promoter and +52 of PAX5beta promoter were significantly associated with lung cancer status (p = 0.0042 and 0.0093, respectively). After adjusting for multiple testing, both loci were of borderline significance (p(adj) = 0.054 and 0.031). (5) The DAPK gene had a regional methylation pattern with two blocks (1) approximately -215--113 and (2) -84-+26; while similar in block 1, there was a significant case-control difference in methylation density in block 2 (p = 0.045); (6)Tumor stage and histology did not impact on the methylation density among the cases. (7) The results of qMSP applied to EBC correlated with the corresponding tBGS sequencing map loci. CONCLUSION: Our results show that DNA methylation in exhaled breath condensate is detectable and is likely of lung origin. Suggestive correlations with smoking and lung cancer case-control status depend on individual gene and CpG site examined.

Investigations of the posttranslational mechanism of arsenite-mediated downregulation of human cytochrome P4501A1 levels: the role of heme oxygenase-1.

Arsenite, an environmental cocontaminant of polycyclic aromatic hydrocarbons (PAHs), diminishes the PAH-mediated upregulation of human CYP1A1, the enzyme that bioactivates PAHs to carcinogenic metabolites. Mechanistically, while transcriptional downregulation contributes to these effects, a role for posttranslational regulation has been implicated but not proven. We hypothesize that arsenite induces heme oxygenase-1 (HO-1), which catabolizes CYP1A1 heme or cellular heme pools, thereby downregulating CYP1A1. Arsenite (5 microM), in HepG2 cells, induced HO-1 mRNA 7.4-fold over the 48 h observation period, and it upregulated HO-1 protein expression. Arsenite decreased the induction of CYP1A1 by a PAH, benzo[k]fluoranthene (BKF), by 50%; and transfection of HepG2 cells with siRNA targeting the human HO-1 gene, reduced the arsenite downregulation of BKF-induced CYP1A1 from 54% to 27%, relative to untransfected cells. Reconstituted HO-1 did not significantly catabolize CYP1A1 heme in vitro. Together these findings demonstrate that a posttranslational mechanism involving decreases in the cellular heme pool by arsenite-induced HO-1 may contribute to arsenite-mediated downregulation of CYP1A1.