Exposure to carbon black nanoparticles during pregnancy aggravates lipopolysaccharide-induced lung injury in offspring: an intergenerational effect

Carbon black nanoparticles (CBNPs) are one of the most frequently used nanoparticles. Exposure to CBNPs during pregnancy (PrE to CBNPs) can directly induce inflammation, lung injury and genotoxicity in dams, and results in abnormalities in offspring. However, whether exposure to CBNPs during pregnancy enhances the susceptibility of offspring to environmental stimuli remains unknown. To address this issue, in this study, we intranasally treated pregnant mice with mock or CBNPs from gestational day (GD) 9 to GD18, and F1 and F2 offspring were normally obtained. By intratracheal instillation of mice with lipopolysaccharide (LPS) to trigger a classic animal model for acute lung injury, we intriguingly found that after LPS treatment, F1 and F2 offspring after exposure during pregnancy to CBNPs both exhibited more pronounced lung injury symptoms, including more degenerative histopathological changes, vascular leakage, elevated MPO activity and activation of inflammation-related signaling transduction, compared to F1 and F2 offspring in the mock treatment group, suggesting PrE to CBNPs would aggravate LPS-induced lung injury in offspring, and this effect is intergenerational. We also observed that PrE to CBNPs upregulated the mRNA expression of DNA methyltransferases (Dnmt) 1/3a/3b and DNA hypermethylation in both F1 and F2 offspring, which might partially account for the intergenerational effect. Together, our study demonstrates for the first time that PrE to CBNPs can enhance sensitivity to LPS in both F1 and F2 offspring, and this intergenerational effect may be related to DNA hypermethylation caused by CBNPs.

Dynamic changes in transposable element and gene methylation in mulberry (Morus notabilis) in response to Botrytis cinerea

DNA methylation has been proposed to regulate plant stress resistance. However, the dynamic changes in DNA methylation in woody plants and their correlations with pathogenic responses are not fully understood. Here, we present single-base maps of the DNA methylomes of mulberry (Morus notabilis) leaves that were subjected to a mock treatment or inoculation with Botrytis cinerea. Compared with the former, the latter showed decreased mCG and mCHG levels and increased mCHH levels. DNA methylation inhibitors reduced resistance gene methylation levels and enhanced mulberry resistance, suggesting that the hypomethylation of resistance genes affects mulberry resistance to B. cinerea. Virus-induced gene silencing of MnMET1 enhanced the expression of mulberry-resistance genes, thereby increasing the plant’s resistance to B. cinerea. We also found that MITEs play a dominant role in controlling DNA methylation levels. MITEs appear to be the main sources of 24-nt siRNAs that regulate gene expression through the RNA-directed DNA methylation pathway.

Quality of Immature and Mature Pepper (Capsicum annuum L.) Seeds in Relation to Bio-Priming with Endophytic Pseudomonas and Bacillus spp.

Fruit maturity for seed production can occur at various times because of the continual flowering of pepper plants. Accordingly, seeds with different maturity are acquired as the fruits are collected in a single harvest. Immature seeds obtained in this harvest may lead to a decrease in the quality of seed lots. Therefore, this research aimed to evaluate the influence of four different endophytic bacteria strains (Pseudomonas fluorescens strain L5b, Pseudomonas gessardii strain L13, Bacillus subtilis strain Bs1 and Bacillus mojavensis strain ApBm) on germination and seedling vigor of immature and mature bell pepper seeds. To obtain seeds with different maturity levels, fruits were collected 45–49 days after flowering for immature seeds and 65–69 days for mature seeds. The effectiveness of these bacteria strains was examined by coating seeds with four different endophytic bacteria strains separately. Additionally, to see the activity of endophytic bacteria more clearly, a mock treatment with sterile water was added to the experiment as a control (+) group. Bio-priming (especially strain Bs1 and L13) improved germination and seedling emergence characteristics of both immature and mature seed lots compared to control groups (p < 0.05). The results demonstrate that bio-priming with beneficial endophytic bacteria can be used to stimulate the quality of both immature and mature seeds from the pepper.

Supplemental Ascorbate Diminishes DNA Damage Yet Depletes Glutathione and Increases Acute Liver Failure in a Mouse Model of Hepatic Antioxidant System Disruption

Cellular oxidants are primarily managed by the thioredoxin reductase-1 (TrxR1)- and glutathione reductase (Gsr)-driven antioxidant systems. In mice having hepatocyte-specific co-disruption of TrxR1 and Gsr (TrxR1/Gsr-null livers), methionine catabolism sustains hepatic levels of reduced glutathione (GSH). Although most mice with TrxR1/Gsr-null livers exhibit long-term survival, ~25% die from spontaneous liver failure between 4- and 7-weeks of age. Here we tested whether liver failure was ameliorated by ascorbate supplementation. Following ascorbate, dehydroascorbate, or mock treatment, we assessed survival, liver histology, or hepatic redox markers including GSH and GSSG, redox enzyme activities, and oxidative damage markers. Unexpectedly, rather than providing protection, ascorbate (5 mg/mL, drinking water) increased the death-rate to 43%. In adults, ascorbate (4 mg/g × 3 days i.p.) caused hepatocyte necrosis and loss of hepatic GSH in TrxR1/Gsr-null livers but not in wildtype controls. Dehydroascorbate (0.3 mg/g i.p.) also depleted hepatic GSH in TrxR1/Gsr-null livers, whereas GSH levels were not significantly affected by either treatment in wildtype livers. Curiously, however, despite depleting GSH, ascorbate treatment diminished basal DNA damage and oxidative stress markers in TrxR1/Gsr-null livers. This suggests that, although ascorbate supplementation can prevent oxidative damage, it also can deplete GSH and compromise already stressed livers.

CRISPR Turbo Accelerated Knock Out (CRISPy TAKO) for rapid in vivo screening of gene function

The development of CRISPR/Cas9 technology has vastly sped up the process of genome editing by introducing a bacterial system that can be exploited for reverse genetics-based research. However, generating homozygous knockout (KO) animals using traditional CRISPR/Cas9-mediated techniques requires three generations of animals. A founder animal with a desired mutation is crossed to produce heterozygous F1 offspring which are subsequently interbred to generate homozygous F2 KO animals. This study describes a novel adaptation of the CRISPR/Cas9-mediated method to develop a homozygous gene-targeted KO animal cohort in one generation. A well-characterized ethanol-responsive gene, MyD88, was chosen as a candidate gene for generation of MyD88-/- mice as proof of concept. Previous studies have reported changes in ethanol-related behavioral outcomes in MyD88 KO mice. Therefore, it was hypothesized that a successful one-generation KO of MyD88 should reproduce decreased responses to ethanols sedative effects, as well as increased ethanol consumption in males that were observed in previous studies. One-cell mouse embryos were simultaneously electroporated with four gRNAs targeting a critical Exon of MyD88 along with Cas9. DNA and RNA analysis of founder mice revealed a complex mix of genetic alterations, all of which were predicted to ablate MyD88 gene function. This study additionally compared responses of Mock treatment control mice generated through electroporation to controls purchased from a vendor. No substantial behavioral changes were noted between control cohorts. Overall, the CRISPR/Cas9 KO protocol reported here, which we call CRISPR Turbo Accelerated KnockOut (CRISPy TAKO), will be useful for reverse genetic in vivo screens of gene function in whole animals.

Comparative proteomic analysis of pepper (Capsicum annuum L.) seedlings under selenium stress

Selenium (Se) is an essential trace element for human and animal health. Se fertilizer has been used to increase the Se content in crops to meet the Se requirements in humans and animals. To address the challenge of Se poisoning in plants, the mechanisms underlying Se-induced stress in plants must be understood. Here, to elucidate the effects of Se stress on the protein levels in pepper, we used an integrated approach involving tandem mass tag labeling, high performance liquid chromatography fractionation, and mass spectrometry-based analysis. A total of 4,693 proteins were identified, 3,938 of which yielded quantitative information. Among them, the expression of 172 proteins was up-regulated, and the expression of 28 proteins was down-regulated in the Se/mock treatment comparison. According to the above data, we performed a systematic bioinformatics analysis of all identified proteins and differentially expressed proteins (DEPs). The DEPs were most strongly associated with the terms “metabolic process,” “posttranslational modification, protein turnover, chaperones,” and “protein processing in endoplasmic reticulum” according to Gene Ontology, eukaryotic orthologous groups classification, and Kyoto Encyclopedia of Genes and Genomes enrichment analysis, respectively. Furthermore, several heat shock proteins were identified as DEPs. These results provide insights that may facilitate further studies on the pepper proteome expressed downstream of the Se stress response. Our data revealed that the responses of pepper to Se stress involve various pathways.

Evaluation of Teratogenic Activity of Antiepileptic Drug Lamotrigine in Mouse Fetuses

Background: Use of antiepileptic drugs during pregnancy can be associated with an increased risk of teratogenicity as well as congenital abnormalities. However, there are numerous discrepancies to determine whether lamotrigine, as an antiepileptic drug, can significantly induce malformation in newborn infants or not. Thus, the purpose of the study was to evaluate the teratogenic effects of lamotrigine on mouse fetuses. Materials and methods: In the present study, 21 pregnant mice were assigned to four groups. Groups 1 and 2 (controls) received mock treatment and ethanol 20%, respectively. Groups 3 and 4 (treatment) were intraperitoneally administered with 25 and 75 mg/kg lamotrigine for three days, respectively. The treatment protocol was performed within the gestational days of 9-18 in all groups. On gestational day 18, 117 fetuses were taken out of the fallopian tube of studied mice and then examined for any anomalies (vertebral, limbs and cranial), followed by a measurement of their height and weight. Results: The results revealed that, in the treated groups, the weight and the height had significantly decreased (p<0.01) and also various anomalies were evident. Moreover, as the dose of lamotrigine increased, the decrease in the weight and the height and rising trend in anomalies were intensified. Conclusion: According to the findings, lamotrigine (LTG) could be considered as a risk factor for the development of the anomalies examined.

Herbicide injury induces DNA methylome alterations in Arabidopsis

The emergence of herbicide-resistant weeds is a major threat facing modern agriculture. Over 470 weedy-plant populations have developed resistance to herbicides. Traditional evolutionary mechanisms are not always sufficient to explain the rapidity with which certain weed populations adapt in response to herbicide exposure. Stress-induced epigenetic changes, such as alterations in DNA methylation, are potential additional adaptive mechanisms for herbicide resistance. We performed methylC sequencing of Arabidopsis thaliana leaves that developed after either mock treatment or two different sub-lethal doses of the herbicide glyphosate, the most-used herbicide in the history of agriculture. The herbicide injury resulted in 9,205 differentially methylated regions (DMRs) across the genome. In total, 5,914 of these DMRs were induced in a dose-dependent manner, wherein the methylation levels were positively correlated to the severity of the herbicide injury, suggesting that plants can modulate the magnitude of methylation changes based on the severity of the stress. Of the 3,680 genes associated with glyphosate-induced DMRs, only 7% were also implicated in methylation changes following biotic or salinity stress. These results demonstrate that plants respond to herbicide stress through changes in methylation patterns that are, in general, dose-sensitive and, at least partially, stress-specific.

Phage Therapy To Reduce Preprocessing Salmonella Infections in Market-Weight Swine

ABSTRACT Contamination of meat products with food-borne pathogens usually results from the carcass coming in contact with the feces of an infected animal during processing. In the case of Salmonella, pigs can become colonized with the organism during transport and lairage from contaminated trailers and holding pens, resulting in increased pathogen shedding just prior to processing. Increased shedding, in turn, amplifies the likelihood of carcass contamination by magnifying the amount of bacteria that enters the processing facility. We conducted a series of experiments to test whether phage therapy could limit Salmonella infections at this crucial period. In a preliminary experiment done with small pigs (3 to 4 weeks old; 30 to 40 lb), administration of an anti-Salmonella phage cocktail at the time of inoculation with Salmonella enterica serovar Typhimurium reduced Salmonella colonization by 99.0 to 99.9% (2- to 3-log reduction) in the tonsils, ileum, and cecum. To test the efficacy of phage therapy in a production-like setting, we inoculated four market-weight pigs (in three replicates) with Salmonella enterica serovar Typhimurium and allowed the challenged pigs to contaminate a holding pen for 48 h. Sixteen naïve pigs were randomly split into two groups which received either the anti-Salmonella phage cocktail or a mock treatment. Both groups of pigs were comingled with the challenged pigs in the contaminated pen. Treatment with the anti-Salmonella phage cocktail significantly reduced cecal Salmonella concentrations (95%; P < 0.05) while also reducing (numerically) ileal Salmonella concentrations (90%; P = 0.06). Additional in vitro studies showed that the phage cocktail was also lytic against several non-Typhimurium serovars.