Microglial NF-κB Signaling Deficiency Protects Against Metabolic Disruptions Caused by Volatile Organic Compound via Modulating the Hypothalamic Transcriptome.

Prolonged exposure to benzene, a prevalent volatile organic compound (VOC), at concentrations found in smoke, triggers hyperglycemia, and inflammation in mice. Corroborating this with existing epidemiological data, we show a strong correlation between environmental benzene exposure and metabolic impairments in humans. To uncover the underlying mechanisms, we employed a controlled exposure system and continuous glucose monitoring (CGM), revealing rapid blood glucose surges and disturbances in energy homeostasis in mice. These effects were attributed to alterations in the hypothalamic transcriptome, specifically impacting insulin and immune response genes, leading to hypothalamic insulin resistance and neuroinflammation. Moreover, benzene exposure activated microglial transcription characterized by heightened expression of IKKß/NF-κB-related genes. Remarkably, selective removal of IKKß in immune cells or adult microglia in mice alleviated benzene-induced hypothalamic gliosis, and protected against hyperglycemia. In summary, our study uncovers a crucial pathophysiological mechanism, establishing a clear link between airborne toxicant exposure and the onset of metabolic diseases.

Thymol-based submicron emulsions exhibit antifungal activity against Fusarium graminearum and inhibit Fusarium head blight in wheat.

AIMS: Fusarium graminearum is a very destructive fungal pathogen that leads to Fusarium head blight (FHB) in wheat, a disease which costs growers millions of dollars annually both in crop losses and in remediation efforts. Current countermeasures include the deployment of wheat varieties with some resistance to FHB in conjunction with timed fungicide treatments. In this article, we introduce a fungicide based on thymol, a naturally occurring plant phenolic derived from essential oils. To overcome the hydrophobicity of thymol, the thymol active was incorporated into a low-surfactant submicron emulsion with and without a carrier oil. METHODS AND RESULTS: The minimum fungicidal concentration of F. graminearum was found to be both 0·02% for thymol emulsions with and without an oil component. Time-to-kill experiments showed that thymol emulsions were able to inactivate F. graminearum in as little as 10 s at concentrations above 0·06%. Spraying the thymol emulsions (~0·1% range) on the wheat variety Bobwhite demonstrated significant reductions in FHB infection rate (number of infected spikelets). However, with 0·5% thymol, the wheat heads exhibited premature senescence. Transmission and scanning electron micrographs suggest that the mechanism of antifungal action is membrane mediated, as conidia exposed to thymol showed complete organelle disorganization and evidence of lipid emulsification. CONCLUSION: The collective experimental data suggest that thymol emulsions may be an effective naturally derived alternative to the current thymol treatments, and chemical fungicides in ameliorating FHB. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first thymol-derived nanoemulsion particles resuspended into water and not DMSO, exhibiting the same antibacterial/antifungal activity as previously described thymol and thyme oil treatments. This drastically reduces the environmental footprint thymol will leave if utilized as a fungicide treatment on field crops.

A 5-year retrospective analysis of 5α-reductase inhibitors in men with benign prostatic hyperplasia: finasteride has comparable urinary symptom efficacy and prostate volume reduction, but less sexual side effects and breast complications than dutasteride.

OBJECTIVE: We evaluated 5-year safety, efficacy and prostate volume data from BPH patients treated with finasteride or dutasteride. METHODS: A retrospective analysis of 378 consecutive men treated with 5α-reductase inhibitor monotherapy between January 2004 and September 2009 (197 on finasteride and 211 on dutasteride) in a single clinic was performed. Efficacy assessments included International Prostate Symptom Score (IPSS), peak urinary flow rate (Qmax), postvoid residual urine volume (PVR), prostate-specific antigen (PSA) and prostate volume (PV). Safety assessments included International Index of Erectile Function (IIEF) and adverse events. Patients were evaluated at 3 months, 1 year and yearly thereafter. RESULTS: Mean age of the group was 58.7 ± 6.7 years. Maintenance of therapy at 5 years was 57.4% and 42.5% for the finasteride and dutasteride groups respectively. Changes in IPSS, Qmax, PVR, PV and PSA were similar for both groups at 5 years. The incidence of erectile dysfunction, ejaculatory dysfunction and decreased libido resulting in discontinuation from therapy was significantly (p < 0.01) higher in the dutasteride (5.1%, 2.4%, 2.7% respectively) compared with the finasteride (2.1%, 1.8%, 1.4% respectively) group. In addition, the incidence of self-reported breast tenderness and/or enlargement was significantly (p < 0.01) greater in the dutasteride (3.5%) compared with the finasteride (1.2%) group. CONCLUSIONS: In this retrospective analysis of data from consecutive patients treated at a single clinic, both finasteride and dutasteride were effective therapies for the management of lower urinary tract symptoms. However, dutasteride resulted in significantly more sexual side effects and breast complications than finasteride.

Screening events to reduce farmers' hazardous exposures.

Dairy farmers may be exposed to high levels of noise and dust. Protections against these hazards exist, but many farmers do not use them. An intervention consisting of hearing and respiratory screenings combined with personalized education was implemented. This study evaluates the impact of this intervention on farmers' self-reported use of personal protective equipment (PPE) and implementation of noise and dust abatement. Participants were screened as to noise (n=209) or dust (n=392) hazards and use of PPE. Following this, they were counseled on PPE use, and identification and reduction of noise or dust hazards. Counselors sought a pledge from the farmers to eliminate hazards and increase PPE use. Farmers were subsequently surveyed and asked whether they had implemented the changes. At baseline, 70% (146/209) of farmers exposed to high levels of noise reported poor use ("sometimes", "rarely", or "never") of hearing protection. Results indicated that two months after intervention, 25.2% (28/111) of these subjects had successfully improved their PPE use. At baseline, 79% (311/392) offarmers reported poor use of respiratory protection, with 27.3% (41/150) showing improvement in PPE use within the same time. Strategies to reduce noise hazards were identified by 92.8% (194/209) of hearing screening attendees; 13.2% (18/136) successfully reduced or removed exposure. These values for dust screening attendees were 98.2% (385/392) and 30.7% (54/176), respectively. Use of this intervention appears to be an effective method for increasing PPE use on the farm. However, it is not effective for reducing noise hazards.

Safety awareness among New York farmers.

BACKGROUND: This study was conducted to assess the health status and safety practices among year-round adult farm workers and residents and included a telephone interview survey of 1,727 persons from 552 farms. METHODS: Logistic regression was used to analyze four safety questions. RESULTS: Among 541 farm owner/operators significant predictors of making substitutions in the use of chemicals and major changes to equipment include younger age, more persons assisting on the farm, and higher gross sales. Having training is associated with having more than a high school education. Among all participants the perception that personal protective equipment are useful is associated with being younger, male, an owner/operator or worker, and having at least a high school education. CONCLUSIONS: These findings suggest that older and less educated farmers should be targeted for health and safety programs.

Tomato Prf is a member of the leucine-rich repeat class of plant disease resistance genes and lies embedded within the Pto kinase gene cluster.

In tomato, resistance to Pseudomonas syringae pv. tomato (Pst) strains expressing the avirulence gene avrPto requires the presence of at least two host genes, designated Pto and Prf. Here we report that Prf encodes a protein with leucine-zipper, nucleotide-binding, and leucine-rich repeat motifs, as are found in a number of resistance gene products from other plants. prf mutant alleles (4) were found to carry alterations within the Prf coding sequence. A genomic fragment containing Prf complemented a prf mutant tomato line both for resistance to Pst strains expressing avrPto and for sensitivity to the insecticide Fenthion. Prf resides in the middle of the Pto gene cluster, 24 kb from the Pto gene and 500 bp from the Fen gene.

Germinal transpositions of the maize element Dissociation from T-DNA loci in tomato.

We have analyzed the pattern of germinal transpositions of artificial Dissociation (Ds) transposons in tomato. T-DNA constructs carrying Ds were transformed into tomato, and the elements were trans-activated by crossing to lines transformed with a stabilized Activator (sAc) that expressed the transposase gene. The sAc T-DNA carried a GUS gene to monitor its segregation. The Ds elements were inserted in a marker gene so that excision from the T-DNA could be monitored. The Ds elements also carried a genetic marker that was intended to be used for reinsertion selection of the elements after excision. Unfortunately, this gene was irreversibly inactivated on crossing to sAc. Germinal excision frequencies of Ds averaged 15-40%, but there was large variation between and within plants. Southern hybridization analysis of stable transposed Ds elements indicated that although unique transpositions predominate, early transposition events can lead to large clonal sectors in the germline of developing plants and to sibling offspring carrying the same transposition event. Multiple germinal transpositions from three different loci were examined for uniqueness, and 15 different transpositions were identified from each of three T-DNA loci that carried a single independent Ds. These were mapped relative to the donor T-DNA loci, and for each locus 70-80% of the transposed elements were closely linked to the donor site.

Modification of the 5' untranslated leader region of the maize Activator element leads to increased activity in Arabidopsis.

In contrast to its behavior in tobacco and tomato, the maize transposable element Ac is relatively inactive in Arabidopsis. We show here that removal of 537 bp within a CpG-rich region of the Ac 5' untranslated leader region significantly increases the excision frequency of the element in Arabidopsis. This increase did not appear to be correlated with the removal of sequences that are methylated in inactive Ac elements in maize, as these sites were not methylated in Ac elements in Arabidopsis transformants. The deletion within the 5' untranslated leader did not increase Ac activity by increasing levels of steady-state transposase mRNA, as assayed by RNase protection experiments. Moreover, there was no correlation between the levels of steady-state transposase mRNA and Ac element activity. This suggests that post-transcriptional regulation of Ac activity occurs in Arabidopsis.

Chloroplast targeting of spectinomycin adenyltransferase provides a cell-autonomous marker for monitoring transposon excision in tomato and tobacco.

Antibiotic resistance genes can act as either cell autonomous or non-cell autonomous genetic markers with which to monitor the excision of plant transposons. To convert spectinomycin resistance from a non-cell autonomous resistance to cell autonomous resistance, a transit peptide for chloroplast localization from a petunia ribulose bisphosphate carboxylase (rbcS) gene was fused in-frame to the aadA gene, which confers spectinomycin and streptomycin resistance. Constructs were generated in which the expression of this chimeric gene was prevented by the presence, in the 5' untranslated leader, of the maize transposons Activator (Ac) or Dissociation (Ds). When progeny of tobacco or tomato plants transformed with these constructs were germinated on spectinomycin-containing medium, germinally revertant and somatically variegated individuals could be distinguished.

High level expression of the Activator transposase gene inhibits the excision of Dissociation in tobacco cotyledons.

A fusion of the strong cauliflower mosaic virus 35S promoter to the Activator (Ac) transposase (TPASE) gene does not trigger excision of Dissociation (Ds) continuously during tobacco cotyledon development, although once activated, the 35S promoter remains active throughout embryogeny. Epistasis studies where 35S:TPASE is in trans with later-acting fusions indicate that transient effectiveness for excision results from this fusion inhibiting its own action and that of other TPASE sources. Inhibition depends on the strength of TPASE expression, since fusions of the 35S promoter to a TPASE cDNA accumulate 30-fold lower amounts of TPASE mRNA than the 35S:TPASE gene fusion and do not inhibit excision. We discuss the role of TPASE levels in the curious relationship between Ac activity and Ac dosage in maize.

Transactivation of Ds elements in plants of lettuce (Lactuca sativa).

The maize transposable element, Activator (Ac), is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. In this paper, we describe somatic and germinal transactivation of Ds by chimeric transposase genes in whole plants. Constructs containing either the Ds element or the Ac transposase open reading frame (ORF) were introduced into lettue. The Ds element was located between either the 35S or the Nos promoter and a chimeric spectinomycin resistance gene (which included a transit peptide), preventing expression of spectinomycin resistance. The genomic coding region of the Ac transposase was expressed from the 35S promoter. Crosses were made between 104 independent R1 plants containing Ds and three independent R1 plants expressing transposase. The excision of Ds in F1 progenies was monitored using a phenotypic assay on spectinomycin-containing medium. Green sectors in one-third of the F1 families indicated transactivation of Ds by the transposase at different developmental stages and at different frequencies in lettuce plants. Excision was confirmed using PCR and by Southern analysis. The lack of green sectors in the majority of F1 families suggest that the majority of T-DNA insertion sites are not conducive to excision. In subsequent experiments, the F1 plants containing both Ds and the transposase were grown to maturity and the F2 seeds screened on medium containing spectinomycin. Somatic excision was again observed in several F2 progeny; however, evidence for germinal excision was observed in only one F2 family.

Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants.

Progress in plant molecular biology has depended heavily on the availability of effective vectors for plant cell transformation and heterologous expression. In this paper we describe the structures of a wide array of plasmids which have proved extremely effective in (a) plant transformation, (b) expression of heterologous genes and (c) assaying the activity of transposons in transgenic plants. Constructs that confer resistance to kanamycin, hygromycin, streptomycin, spectinomycin and phosphinotricin, or that confer beta-glucuronidase (GUS) gene expression are presented. Binary vector constructs that carry polylinkers of the pUC and Bluescript types are also described. Plasmids that permit the expression of any heterologous reading frame from either nopaline synthase (nos) or octopine synthase (ocs) promoters, as well as the cauliflower mosaic virus 35S promoter, using either the nopaline synthase or octopine synthase 3' polyadenylation sequences, are presented. These constructs permit a choice of orientation of the resulting transgene of interest, relative to the orientation of the selection marker gene. Most of the plasmids described here are publicly available.

Development of an efficient two-element transposon tagging system in Arabidopsis thaliana.

Modified Ac and Ds elements, in combination with dominant markers (to facilitate monitoring of excision, reinsertion and segregation of the elements) were introduced into Arabidopsis thaliana ecotype Landsberg erecta. The frequencies of somatic and germinal transactivation of the Ds elements were monitored using a streptomycin resistance assay. Transactivation was significantly higher from a stable Ac (sAc) carrying a 537 bp deletion of the CpG-rich 5' untranslated leader of the transposase mRNA than from a wild-type sAc. However, substitution of the central 1.77 kb of the transposase open reading frame (ORF) with a hygromycin resistance marker did not alter the excision frequency of a Ds element. beta-Glucuronidase (GUS) or iaaH markers were linked to the transposase source to allow the identification of plants in which the transposase source had segregated away from the transposed Ds element, eliminating the possibility of somatic or germinal re-activation. Segregation of the excision marker, Ds and sAc was monitored in the progeny of plants showing germinal excision of Ds. 29% of the plants inheriting the excision marker carried a transposed Ds element.

Promoter fusions to the Activator transposase gene cause distinct patterns of Dissociation excision in tobacco cotyledons.

To explore the effects of altering the level of Activator (Ac) transposase (TPase) expression, a series of plasmids was constructed in which heterologous promoters were fused to the TPase gene. Promoters for the cauliflower mosaic virus (CaMV) 35S transcript and the octopine synthase (ocs) and nopaline synthase (nos) genes were tested. These fusions, and constructs expressing TPase from the wild-type Ac promoter, were introduced into tobacco, and their activity was monitored by crossing to a line carrying Dissociation (Ds) in a streptomycin phosphotransferase gene (Ds::SPT). The SPT marker provides a record of somatic excisions of Ds that occur during embryo development. The patterns of somatic variegation that resulted from transactivation by each fusion were distinct and strikingly different from the pattern triggered by the wild-type Ac constructs. Unlike wild-type Ac, which caused transposition throughout embryo development, each fusion gave rise to sectors of discrete size. Sectors triggered by the CaMV 35S fusion were largest, ocs sectors were intermediate, and nos were smallest. These patterns appear to indicate differential timing of the activation of these promoters during embryogeny. Measurement of transcript abundance for each transformant indicated that the CaMV 35S-transformed plants accumulated approximately 1000-fold more TPase mRNA than plants containing wild-type Ac, whereas ocs- and nos-transformed lines accumulated about 100-fold and 20-fold higher levels, respectively. Measurements of germinal excision frequencies driven by the chimeric TPase fusions, however, indicated that increasing transcription does not necessarily result in an increase in germinal excision. These measurements showed that the ocs and nos fusions have very low rates of germinal excision. Only the CaMV 35S fusion transformants were found to have higher rates than the Ac constructs, although significant pod-to-pod variation was observed. Gel blot analysis of DNA from progeny carrying germinal excision events resulting from the CaMV 35S fusion showed that excision is associated with reinsertion and that siblings sometimes carry the same transposition events. These findings suggest that in tobacco there is no direct proportionality between TPase expression and Ac-Ds transposition activity. This possibility has important implications for understanding the regulation of Ac transposition and for designing efficient gene tagging systems.

Elevated levels of Activator transposase mRNA are associated with high frequencies of Dissociation excision in Arabidopsis.

The Activator (Ac) element of maize is active at a low frequency in Arabidopsis. To determine whether this is due to poor expression of the Ac transposase gene, we obtained and studied 19 Arabidopsis transformants containing fusions of the octopine synthase (ocs), nopaline synthase (nos), cauliflower mosaic virus (CaMV) 35S, or Ac promoters to the transposase open reading frame. These transformants were examined both for their ability to drive excision of a Dissociation (Ds) element from a streptomycin resistance gene and for the abundance of the transposase mRNA. Most transformants containing the CaMV 35S fusion have high levels of transposase transcript and drive high frequencies of somatic and germinal excision. These results demonstrated that Arabidopsis contains all of the host functions required for high frequency excision of Ds. Moreover, transposase mRNA abundance varied about 1000-fold among our transformants; this variation enabled us to demonstrate that for the Ac, ocs, and CaMV 35S fusion, raising the mRNA level is closely correlated with increasing excision frequency. We discuss our data in relation to the behavior of Ac in Arabidopsis, maize, and tobacco.

Novel DNA structures resulting from dTam3 excision in tobacco.

A Tam3 two-element system has been designed by combining an immobilized Tam3 element with a non-autonomous dTam3 element inserted into the HPT gene. The phenotypic assay employed, restored hygromycin resistance, indicated that trans-activation of the non-autonomous dTam3 element occurred. Molecular analyses of the excision sites revealed that the ends of the dTam3 element remain in the empty donor sites. The predominant consequence of this type of excision appears to be that excised fragments fail to re-integrate into the tobacco genome. Only one case of dTam3 re-integration could be detected. The ends of this element had been degraded upon integration into the tobacco genome. Either the altered structure of the Tam3 derivatives or tobacco host factors are influencing the trans-activation of a dTam3 element, resulting in aberrant excision.

Delayed Leaf Senescence in Tobacco Plants Transformed with tmr, a Gene for Cytokinin Production in Agrobacterium.

The aim of this study was to investigate whether enhanced levels of endogenous cytokinins could influence plant development, particularly leaf senescence. Tobacco plants were transformed with the Agrobacterium tumefaciens gene tmr, under the control of the soybean heat shock promoter HS6871. This gene encodes the enzyme isopentenyl transferase, which catalyzes the initial step in cytokinin biosynthesis. After heat shock, the cytokinin level increased greatly and the level of tmr mRNA, undetectable at 20[deg]C, rose and remained high for up to 8 hours. The levels of cytokinin and tmr mRNA were substantially lower by 24 hours. Transformed plants grown at 20[deg]C were shorter, had larger side shoots, and remained green for longer than untransformed plants. The differences were more pronounced after several heat shocks of whole plants or defined areas of leaves. Our results demonstrated that plant morphology and leaf senescence can be manipulated by changing the endogenous level of cytokinins.

Development of an efficient transposon tagging system in Arabidopsis thaliana.

We are currently developing a transposon tagging system in Arabidopsis thaliana using the maize transposable elements Ac and Ds. In order to make the system as efficient as possible, four different antibiotic resistance markers have been tested for their usefulness in monitoring excision and reinsertion of transposons in the Arabidopsis genome. Owing to the low transposition frequency of wild-type Ac in Arabidopsis we have also tested a number of modifications to the Ac element. Deletion of the CpG-rich region in the transposase 5' untranslated leader was found to significantly increase the activity of Ac in Arabidopsis. In our first non-targeted tagging experiment, 200 individuals with an inherited transposed Ac element have been collected and their progeny are being screened in families of twelve for segregation of mutant phenotypes. A two-element system, using Ac and Ds, is also being developed and a way of stabilising the Ds-induced mutations by counter selecting plants carrying the transposase source is being investigated. The Agrobacterium T-DNA insertions carrying the different transposons are currently being mapped onto the Arabidopsis RFLP map. Inverse polymerase chain reaction (IPCR) is being used to generate flanking DNA probes, that will be used on the RFLP blots to map the T-DNA relative to the other known markers. Once the first few have been mapped the first targeted tagging experiment will be initiated.