Composting reduces the risks of resistome in beef cattle manure at the transcriptional level.

Transcriptomic evidence is needed to determine whether composting is more effective than conventional stockpiling in mitigating the risk of resistome in livestock manure. The objective of this study is to compare composting and stockpiling for their effectiveness in reducing the risk of antibiotic resistance in beef cattle manure. Samples collected from the center and the surface of full-size manure stockpiling and composting piles were subject to metagenomic and metatranscriptomic analyses. While the distinctions in resistome between stockpiled and composted manure were not evident at the DNA level, the advantages of composting over stockpiling were evident at the transcriptomic level in terms of the abundance of antibiotic resistance genes (ARGs), the number of ARG subtypes, and the prevalence of high-risk ARGs (i.e., mobile ARGs associated with zoonotic pathogens). DNA and transcript contigs show that the pathogen hosts of high-risk ARGs included Escherichia coli O157:H7 and O25b:H4, Klebsiella pneumoniae, and Salmonella enterica. Although the average daily temperatures for the entire composting pile exceeded 55°C throughout the field study, more ARG and ARG transcripts were removed at the center of the composting pile than at the surface. This work demonstrates the advantage of composting over stockpiling in reducing ARG risk in active populations in beef cattle manure.IMPORTANCEProper treatment of manure before land application is essential to mitigate the spread of antibiotic resistance in the environment. Stockpiling and composting are two commonly used methods for manure treatment. However, the effectiveness of composting in reducing antibiotic resistance in manure has been debated. This work compared the ability of these two methods to reduce the risk of antibiotic resistance in beef cattle manure. Our results demonstrate that composting reduced more high-risk resistance genes at the transcriptomic level in cattle manure than conventional stockpiling. This finding not only underscores the effectiveness of composting in reducing antibiotic resistance in manure but also highlights the importance of employing RNA analyses alongside DNA analyses.

Porcine Reproductive and Respiratory Syndrome Virus Infection Upregulates Negative Immune Regulators and T-Cell Exhaustion Markers.

Porcine alveolar macrophage (PAM) is one of the primary cellular targets for porcine reproductive and respiratory syndrome virus (PRRSV), but less than 2% of PAMs are infected with the virus during the acute stage of infection. To comparatively analyze the host transcriptional response between PRRSV-infected PAMs and bystander PAMs that remained uninfected but were exposed to the inflammatory milieu of an infected lung, pigs were infected with a PRRSV strain expressing green fluorescent protein (PRRSV-GFP), and GFP+ (PRRSV infected) and GFP- (bystander) cells were sorted for RNA sequencing (RNA-seq). Approximately 4.2% of RNA reads from GFP+ and 0.06% reads from GFP- PAMs mapped to the PRRSV genome, indicating that PRRSV-infected PAMs were effectively separated from bystander PAMs. Further analysis revealed that inflammatory cytokines, interferon-stimulated genes, and antiviral genes were highly upregulated in GFP+ compared to GFP- PAMs. Importantly, negative immune regulators, including NF-κB inhibitors (NFKBIA, NFKBID, NFKBIZ, and TNFAIP3) and T-cell exhaustion markers (programmed death ligand-1 [PD-L1], PD-L2, interleukin-10 [IL-10], IDO1, and transforming growth factor ß2 [TGFB2]) were highly upregulated in GFP+ cells compared to GFP- cells. By using an in situ hybridization assay, RNA transcripts of tumor necrosis factor (TNF) and NF-κB inhibitors were detected in PRRSV-infected PAMs cultured ex vivo and lung sections of PRRSV-infected pigs during the acute stage of infection. Collectively, the results suggest that PRRSV infection upregulates expression of negative immune regulators and T-cell exhaustion markers in PAMs to modulate the host immune response. Our findings provide further insight into PRRSV immunopathogenesis. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is widespread in many swine-producing countries, causing substantial economic losses to the swine industry. Porcine alveolar macrophage (PAM) is considered the primary target for PRRSV replication in pigs. However, less than 2% of PAMs from acutely infected pigs are infected with the virus. In the present study, we utilized a PRRSV strain expressing green fluorescent protein to infect pigs and sorted infected and bystander PAMs from the pigs during the acute stage of infection for transcriptome analysis. PRRSV-infected PAMs showed a distinctive gene expression profile and contained many uniquely activated pathways compared to bystander PAMs. Interestingly, upregulated expression of NF-κB signaling inhibitors and T-cell exhaustion molecules were observed in PRRSV-infected PAMs. Our findings provide additional knowledge on the mechanisms that PRRSV employs to modulate the host immune system.

Effects of Nutrient Level and Growth Rate on the Conjugation Process That Transfers Mobile Antibiotic Resistance Genes in Continuous Cultures.

Bacteria in the effluent of wastewater treatment plants (WWTPs) can transfer antibiotic resistance genes (ARGs) to the bacteria in receiving water through conjugation; however, there is a lack of quantitative assessment of this phenomenon in continuous cultures. Our objective was to determine the effects of background nutrient levels in river water column and growth rates of bacteria on the conjugation frequency of ARGs from effluent bacteria to river bacteria, as well as on the resulting resistance level (i.e., MICs) of the river bacteria. Chemostats were employed to simulate the discharge points of WWTPs into rivers, where effluent bacteria (donor cells) meet river bacteria (recipient cells). Both donor and recipient cells were Escherichia coli cells, and the donor cells were constructed by filter mating with bacteria in the effluent of a local WWTP. Results showed that higher bacterial growth rate (0.45 h-1 versus 0.15 h-1) led to higher conjugation frequencies (10-4 versus 10-6 transconjugant per recipient). The nutrient level also significantly affected the conjugation frequency, albeit to a lesser extent than the growth rate. The MIC against tetracycline increased from 2 mg/L in the recipient to 64 to 128 mg/L in transconjugants. In comparison, the MIC only increased to as high as 8 mg/L in mutants. Whole-genome sequencing showed that the tet-containing plasmid in both the donor and the transconjugant cells also occur in other fecal bacterial genera. The quantitative information obtained from this study can inform hazard identification related to the proliferation of wastewater-associated ARGs in surface water. IMPORTANCE WWTPs have been regarded as an important hot spot of ARGs. The discharge point of WWTP effluent, where ARGs may be horizontally transferred from bacteria of treated wastewater to bacteria of receiving water, is an important interface between the human-dominated ecosystem and the natural environment. The use of batch cultures in previous studies cannot adequately simulate the nutrient conditions and growth rates in receiving water. In this study, chemostats were employed to simulate the continuous growth of bacteria in receiving water. Furthermore, the experimental setup allowed for separate investigations on the effects of nutrient levels (i.e., simulating background nutrients in river water) and bacterial growth rates on conjugation frequencies and resulting resistance levels. The study generates statistically sound ecological data that can be used to estimate the risk of wastewater-originated ARGs as part of the One Health framework.

Natural variation in root exudation of GABA and DIMBOA impacts the maize root endosphere and rhizosphere microbiomes.

Root exudates are important for shaping root-associated microbiomes. However, studies on a wider range of metabolites in exudates are required for a comprehensive understanding about their influence on microbial communities. We identified maize inbred lines that differ in exudate concentrations of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and γ-aminobutyric acid (GABA) using a semi-hydroponic system. These lines were grown in the field to determine the changes in microbial diversity and gene expression due to varying concentrations of DIMBOA and GABA in exudates using 16S rRNA amplicon sequencing and metatranscriptomics. Results showed individual and interaction effects of DIMBOA and GABA on the rhizosphere and root endosphere ß-diversity, most strongly at the V10 growth stage. The main bacterial families affected by both compounds were Ktedonobacteraceae and Xanthomonadaceae. Higher concentrations of DIMBOA in exudates affected the rhizosphere metatranscriptome, enriching for metabolic pathways associated with plant disease. This study validated the use of natural variation within plant species as a powerful approach for understanding the role of root exudates on microbiome selection. We also showed that a semi-hydroponic system can be used to identify maize genotypes that differ in GABA and DIMBOA exudate concentrations under field conditions. The impact of GABA exudation on root-associated microbiomes is shown for the first time.

Transcriptomics of tapping and healing process in frankincense tree during resin production.

Frankincense tree (Boswellia sacra Fluek) has been poorly known on how it responds to tapping and wound-recovery process at molecular levels. Here, we used RNA-sequencing analysis to profile transcriptome of B. sacra after 30 min, 3 h and 6 h of post-tapping. Results showed 5525 differentially expressed genes (DEGs) that were related to terpenoid biosynthesis, phytohormonal regulation, cellular transport, and cell-wall synthesis. Plant-growth-regulators were applied exogenously which showed regulation of endogenous jasmonates and resulted in rapid recovery of cell-wall integrity by significantly up-regulated gene expression of terpenoid biosynthesis (germacrene-D synthase, B-amyrin synthase, and squalene epioxidase-1) and cell-wall synthesis (xyloglucan endotransglucosylase, cellulose synthase-A, and cell-wall hydrolase) compared to control. These findings suggest that tapping immediately activated several cell-developmental and regeneration processes, alongwith defense-induced terpenoid metabolism, to improve the healing process in epidermis. Exogenous growth regulators, especially jasmonic acid, can drastically help tree recovery from tissue degeneration and might help in tree conservation purposes.

Epitope Mapping of SERCA2a Identifies an Antigenic Determinant That Induces Mainly Atrial Myocarditis in A/J Mice.

Sarcoplasmic/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971-990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a 971-990-induced disease was associated with the induction of CD4 T cell responses and the epitope preferentially binds MHC class II/IAk rather than IEk By creating IAk/and IEk/SERCA2a 971-990 dextramers, the T cell responses were determined by flow cytometry to be Ag specific. 3) SERCA2a 971-990-sensitized T cells produce both Th1 and Th17 cytokines. 4) Animals immunized with SERCA2a 971-990 showed Ag-specific Abs with enhanced production of IgG2a and IgG2b isotypes, suggesting that SERCA2a 971-990 can potentially act as a common epitope for both T cells and B cells. 5) Finally, SERCA2a 971-990-sensitized T cells were able to transfer disease to naive recipients. Together, these data indicate that SERCA2a is a critical autoantigen in the mediation of atrial inflammation in mice and that our model may be helpful to study the inflammatory events that underlie the development of conditions such as atrial fibrillation in humans.

Role of whole grains versus fruits and vegetables in reducing subclinical inflammation and promoting gastrointestinal health in individuals affected by overweight and obesity: a randomized controlled trial.

BACKGROUND: Whole grains (WG) and fruits and vegetables (FV) have been shown to reduce the risk of metabolic disease, possibly via modulation of the gut microbiota. The purpose of this study was to determine the impact of increasing intake of either WG or FV on inflammatory markers and gut microbiota composition. METHODS: A randomized parallel arm feeding trial was completed on forty-nine subjects with overweight or obesity and low intakes of FV and WG. Individuals were randomized into three groups (3 servings/d provided): WG, FV, and a control (refined grains). Stool and blood samples were collected at the beginning of the study and after 6 weeks. Inflammatory markers [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), lipopolysaccharide binding protein (LBP), and high sensitivity C-reactive protein (hs-CRP)] were measured. Stool sample analysis included short/branched chain fatty acids (S/BCFA) and microbiota composition. RESULTS: There was a significant decrease in LBP for participants on the WG (- 0.2 µg/mL, p = 0.02) and FV (- 0.2 µg/mL, p = 0.005) diets, with no change in those on the control diet (0.1 µg/mL, p = 0.08). The FV diet induced a significant change in IL-6 (- 1.5 pg/mL, p = 0.006), but no significant change was observed for the other treatments (control, - 0.009 pg/mL, p = 0.99; WG, - 0.29, p = 0.68). The WG diet resulted in a significant decrease in TNF-α (- 3.7 pg/mL; p < 0.001), whereas no significant effects were found for those on the other diets (control, - 0.6 pg/mL, p = 0.6; FV, - 1.4 pg/mL, p = 0.2). The treatments induced individualized changes in microbiota composition such that treatment group differences were not identified, except for a significant increase in α-diversity in the FV group. The proportions of Clostridiales (Firmicutes phylum) at baseline were correlated with the magnitude of change in LBP during the study. CONCLUSIONS: These data demonstrate that WG and FV intake can have positive effects on metabolic health; however, different markers of inflammation were reduced on each diet suggesting that the anti-inflammatory effects were facilitated via different mechanisms. The anti-inflammatory effects were not related to changes in gut microbiota composition during the intervention, but were correlated with microbiota composition at baseline. TRIAL REGISTRATION: ClinicalTrials.gov , NCT02602496 , Nov 4, 2017.

Osmotic stress induces phosphorylation of histone H3 at threonine 3 in pericentromeric regions of Arabidopsis thaliana.

Histone phosphorylation plays key roles in stress-induced transcriptional reprogramming in metazoans but its function(s) in land plants has remained relatively unexplored. Here we report that an Arabidopsis mutant defective in At3g03940 and At5g18190, encoding closely related Ser/Thr protein kinases, shows pleiotropic phenotypes including dwarfism and hypersensitivity to osmotic/salt stress. The double mutant has reduced global levels of phosphorylated histone H3 threonine 3 (H3T3ph), which are not enhanced, unlike the response in the wild type, by drought-like treatments. Genome-wide analyses revealed increased H3T3ph, slight enhancement in trimethylated histone H3 lysine 4 (H3K4me3), and a modest decrease in histone H3 occupancy in pericentromeric/knob regions of wild-type plants under osmotic stress. However, despite these changes in heterochromatin, transposons and repeats remained transcriptionally repressed. In contrast, this reorganization of heterochromatin was mostly absent in the double mutant, which exhibited lower H3T3ph levels in pericentromeric regions even under normal environmental conditions. Interestingly, within actively transcribed protein-coding genes, H3T3ph density was minimal in 5' genic regions, coincidental with a peak of H3K4me3 accumulation. This pattern was not affected in the double mutant, implying the existence of additional H3T3 protein kinases in Arabidopsis. Our results suggest that At3g03940 and At5g18190 are involved in the phosphorylation of H3T3 in pericentromeric/knob regions and that this repressive epigenetic mark may be important for maintaining proper heterochromatic organization and, possibly, chromosome function(s).

Identification of an Epitope from Adenine Nucleotide Translocator 1 That Induces Inflammation in Heart in A/J Mice.

Heart failure, a leading cause of death in humans, can emanate from myocarditis. Although most individuals with myocarditis recover spontaneously, some develop chronic dilated cardiomyopathy. Myocarditis may result from both infectious and noninfectious causes, including autoimmune responses to cardiac antigens. In support of this notion, intracellular cardiac antigens, like cardiac myosin heavy chain-α, cardiac troponin-I, and adenine nucleotide translocator 1 (ANT1), have been identified as autoantigens in cardiac autoimmunity. Herein, we demonstrate that ANT1 can induce autoimmune myocarditis in A/J mice by generating autoreactive T cells. We show that ANT1 encompasses multiple immunodominant epitopes (namely, ANT1 21-40, ANT1 31-50, ANT1 171-190, and ANT1 181-200). Although all four peptides induce comparable T-cell responses, only ANT1 21-40 was found to be a major myocarditogenic epitope in immunized animals. The myocarditis-inducing ability of ANT1 21-40 was associated with the generation of T cells producing predominantly IL-17A, and the antigen-sensitized T cells could transfer the disease to naïve recipients. These data indicate that cardiac mitochondrial proteins can be target autoantigens in myocarditis, supporting the notion that the antigens released as a result of primary damage may contribute to the persistence of chronic inflammation through autoimmunity.

Overaccumulation of γ-Glutamylcysteine in a Jasmonate-Hypersensitive Arabidopsis Mutant Causes Jasmonate-Dependent Growth Inhibition.

Glutathione (GSH) is essential for many aspects of plant biology and is associated with jasmonate signaling in stress responses. We characterized an Arabidopsis (Arabidopsis thaliana) jasmonate-hypersensitive mutant (jah2) with seedling root growth 100-fold more sensitive to inhibition by the hormone jasmonyl-isoleucine than the wild type. Genetic mapping and genome sequencing determined that the mutation is in intron 6 of GLUTATHIONE SYNTHETASE2, encoding the enzyme that converts γ-glutamylcysteine (γ-EC) to GSH. The level of GSH in jah2 was 71% of the wild type, while the phytoalexin-deficient2-1 (pad2-1) mutant, defective in GSH1 and having only 27% of wild-type GSH level, was not jasmonate hypersensitive. Growth defects for jah2, but not pad2, were also seen in plants grown to maturity. Surprisingly, all phenotypes in the jah2 pad2-1 double mutant were weaker than in jah2. Quantification of γ-EC indicated these defects result from hyperaccumulation of this GSH precursor by 294- and 65-fold in jah2 and the double mutant, respectively. γ-EC reportedly partially substitutes for loss of GSH, but growth inhibition seen here was likely not due to an excess of total glutathione plus γ-EC because their sum in jah2 pad2-1 was only 16% greater than in the wild type. Further, the jah2 phenotypes were lost in a jasmonic acid biosynthesis mutant background, indicating the effect of γ-EC is mediated through jasmonate signaling and not as a direct result of perturbed redox status.

Dehydration stress memory genes of Zea mays; comparison with Arabidopsis thaliana.

BACKGROUND: Pre-exposing plants to diverse abiotic stresses may alter their physiological and transcriptional responses to a subsequent stress, suggesting a form of "stress memory". Arabidopsis thaliana plants that have experienced multiple exposures to dehydration stress display transcriptional behavior suggesting "memory" from an earlier stress. Genes that respond to a first stress by up-regulating or down-regulating their transcription but in a subsequent stress provide a significantly different response define the 'memory genes' category. Genes responding similarly to each stress form the 'non-memory' category. It is unknown whether such memory responses exists in other Angiosperm lineages and whether memory is an evolutionarily conserved response to repeated dehydration stresses. RESULTS: Here, we determine the transcriptional responses of maize (Zea mays L.) plants that have experienced repeated exposures to dehydration stress in comparison with plants encountering the stress for the first time. Four distinct transcription memory response patterns similar to those displayed by A. thaliana were revealed. The most important contribution is the evidence that monocot and eudicot plants, two lineages that have diverged 140 to 200 M years ago, display similar abilities to 'remember' a dehydration stress and to modify their transcriptional responses, accordingly. The highly sensitive RNA-Seq analyses allowed to identify genes that function similarly in the two lineages, as well as genes that function in species-specific ways. Memory transcription patterns indicate that the transcriptional behavior of responding genes under repeated stresses is different from the behavior during an initial dehydration stress, suggesting that stress memory is a complex phenotype resulting from coordinated responses of multiple signaling pathways. CONCLUSIONS: Structurally related genes displaying the same memory responses in the two species would suggest conservation of the genes' memory during the evolution of plants' dehydration stress response systems. On the other hand, divergent transcription memory responses by genes encoding similar functions would suggest occurrence of species-specific memory responses. The results provide novel insights into our current knowledge of how plants respond to multiple dehydration stresses, as compared to a single exposure, and may serve as a reference platform to study the functions of memory genes in adaptive responses to water deficit in monocot and eudicot plants.

The impact of iron supplementation on the preterm neonatal gut microbiome: A pilot study.

OBJECTIVE: The gastrointestinal microbiome in preterm infants exhibits significant influence on optimal outcomes-with dysbiosis shown to substantially increase the risk of the life-threatening necrotizing enterocolitis. Iron is a vital nutrient especially during the perinatal window of rapid hemoglobin production, tissue growth, and foundational neurodevelopment. However, excess colonic iron exhibits potent oxidation capacity and alters the gut microbiome-potentially facilitating the proliferation of pathological bacterial strains. Breastfed preterm infants routinely receive iron supplementation starting 14 days after delivery and are highly vulnerable to morbidities associated with gastrointestinal dysbiosis. Therefore, we set out to determine if routine iron supplementation alters the preterm gut microbiome. METHODS: After IRB approval, we collected stool specimens from 14 infants born <34 weeks gestation in the first, second, and fourth week of life to assess gut microbiome composition via 16S rRNA sequencing. RESULTS: We observed no significant differences in either phyla or key genera relative abundance between pre- and post-iron timepoints. We observed notable shifts in infant microbiome composition based on season of delivery. CONCLUSION: Though no obvious indication of iron-induced dysbiosis was observed in this unique study in the setting of prematurity, further investigation in a larger sample is warranted to fully understand iron's impact on the gastrointestinal milieu.

Liver matrin-3 protects mice against hepatic steatosis and stress response via constitutive androstane receptor.

OBJECTIVE: The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) continues to rise with the increasing obesity epidemic. Rezdiffra as an activator of a thyroid hormone receptor-beta is the only Food and Drug Administration approved therapy. As such, there is a critical need to improve our understanding of gene expression regulation and signaling transduction in MASLD to develop new therapies. Matrin-3 is a DNA- and RNA-binding protein involved in the pathogenesis of human diseases. Here we examined its previously uncharacterized role in limiting hepatic steatosis and stress response via the constitutive androstane receptor (CAR). METHODS: Matrin-3 floxed and liver-specific knockout mice were fed either a chow diet or 60 kcal% high-fat diet (HFD) for up to 16 weeks. The mice were euthanized for different analysis including liver histology, lipid levels, and gene expression. Bulk RNA-seq, bulk ATAC-seq, and single-nucleus Multiome were used to examine changes of transcriptome and chromatin accessibility in the liver. Integrative bioinformatics analysis of our data and publicly available datasets and different biochemical assays were performed to identify underlying the molecular mechanisms mediating matrin-3's effects. Liver-tropic adeno-associated virus was used to restore the expression of CAR for lipid, acute phase genes, and histological analysis. RESULTS: Matrin-3 expression is induced in the steatotic livers of mice. Liver-specific matrin-3 deletion exacerbated HFD-induced steatosis, acute phase response, and inflammation in the liver of female mice. The transcriptome and chromatin accessibility were re-programmed in the liver of these mice with signatures indicating that CAR signaling is dysregulated. Mechanistically, matrin-3 interacts with CAR mRNA, and matrin-3 deficiency promotes CAR mRNA degradation. Consequently, matrin-3 deletion impaired CAR signaling by reducing CAR expression. Matrin-3 levels positively correlate with CAR expression in human livers. Ces2a and Il1r1 were identified as new target genes of CAR. Interestingly, we found that CAR discords with the expression of its target genes including Cyp2b10 and Ces2a in response to HFD, indicating CAR signaling is dysregulated by HFD despite increased CAR expression. Dysregulated CAR signaling upon matrin-3 deficiency reduced Ces2a and de-repressed Il1r1 expression. CAR restoration partially abrogated the dysregulated gene expression, exacerbated hepatic steatosis, acute phase response, and inflammation in liver-specific matrin-3 knockout mice fed a HFD. CONCLUSIONS: Our findings demonstrate that matrin-3 is a key upstream regulator maintaining CAR signaling upon metabolic stress, and the matrin-3-CAR axis limits hepatic steatosis and stress response signaling that may give insights for therapeutic intervention.

Characterization of genome-wide transcriptional changes in liver and adipose tissues of ZDF (fa/fa) rats fed R-α-lipoic acid by next-generation sequencing.

We report on the characterization of lipogenic tissue transcriptional networks that support physiological responses of obese rats to a lipid-lowering bioactive food compound, R-α-lipoic acid (LA). Nine-week-old male Zucker diabetic fatty (fa/fa) rats were fed a chow diet supplemented with 3 g LA per kg diet or pair fed for 2 wk. At the end of the trial, high-quality RNA was extracted from the liver and epididymal fat and subjected to transcriptome analysis by RNA-Seq technology. Results showed a substantially higher number of differentially expressed genes [DEG, false discovery rate adjusted P ≤ 0.05 and absolute log2 (fold change) ≥ 1] in the liver (110 genes) vs. epididymal fat (10 genes). Most epididymal fat DEG were also differentially expressed in liver and shared directionality of change. Gene Ontology (GO) analysis of these transcripts revealed significant enrichment of GO categories related to immune response, stress response, lipid metabolism, and carboxylic acid metabolic processes. Of interest, interferon-related genes involved in defense against microorganisms and innate immune response were induced by LA. Lipid metabolism-related transcript changes observed in LA-fed animals included downregulation of lipogenic genes (Pnpla3, Pnpla5, Elovl6, Acly, Gpam, and Aacs) and concomitant upregulation of short-, medium-, and long-chain fatty acid metabolic processes (Acot1, Acot2, Acsf2, and Crat). Transcriptional changes were accompanied by the lowering of abdominal adiposity and blood triacylglycerol levels. We conclude that LA dietary supplementation induces prominent gene expression changes in liver in support of significant improvement of whole-body lipid status.

Divergent functions of the myotubularin (MTM) homologs AtMTM1 and AtMTM2 in Arabidopsis thaliana: evolution of the plant MTM family.

Myotubularin and myotubularin-related proteins are evolutionarily conserved in eukaryotes. Defects in their function result in muscular dystrophy, neuronal diseases and leukemia in humans. In contrast to the animal lineage, where genes encoding both active and inactive myotubularins (phosphoinositide 3-phosphatases) have appeared and proliferated in the basal metazoan group, myotubularin genes are not found in the unicellular relatives of green plants. However, they are present in land plants encoding proteins highly similar to the active metazoan enzymes. Despite their remarkable structural conservation, plant and animal myotubularins have significantly diverged in their functions. While loss of myotubularin function causes severe disease phenotypes in humans it is not essential for the cellular homeostasis under normal conditions in Arabidopsis thaliana. Instead, myotubularin deficiency is associated with altered tolerance to dehydration stress. The two Arabidopsis genes AtMTM1 and AtMTM2 have originated from a segmental chromosomal duplication and encode catalytically active enzymes. However, only AtMTM1 is involved in elevating the cellular level of phosphatidylinositol 5-phosphate in response to dehydration stress, and the two myotubularins differentially affect the Arabidopsis dehydration stress-responding transcriptome. AtMTM1 and AtMTM2 display different localization patterns in the cell, consistent with the idea that they associate with different membranes to perform specific functions. A single amino acid mutation in AtMTM2 (L250W) results in a dramatic loss of subcellular localization. Mutations in this region are linked to disease conditions in humans.

Four distinct types of dehydration stress memory genes in Arabidopsis thaliana.

BACKGROUND: How plants respond to dehydration stress has been extensively researched. However, how plants respond to multiple consecutive stresses is virtually unknown. Pre-exposure to various abiotic stresses (including dehydration) may alter plants' subsequent responses by improving resistance to future exposures. These observations have led to the concept of 'stress memory' implying that during subsequent exposures plants provide responses that are different from those during their first encounter with the stress. Genes that provide altered responses in a subsequent stress define the 'memory genes' category; genes responding similarly to each stress form the 'non-memory' category. RESULTS: Using a genome-wide RNA-Seq approach we determine the transcriptional responses of Arabidopsis plants that have experienced multiple exposures to dehydration stress and compare them with the transcriptional behavior of plants encountering the stress for the first time. The major contribution of this study is the revealed existence of four distinct, previously unknown, transcription memory response patterns of dehydration stress genes in A.thaliana. The biological relevance for each of the four memory types is considered in the context of four overlapping strategies employed by a plant to improve its stress tolerance and/or survival: 1) increased synthesis of protective, damage-repairing, and detoxifying functions; 2) coordinating photosynthesis and growth under repetitive stress; 3) re-adjusting osmotic and ionic equilibrium to maintain homeostasis; and 4) re-adjusting interactions between dehydration and other stress/hormone regulated pathways. CONCLUSIONS: The results reveal the unknown, hitherto, existence of four distinct transcription memory response types in a plant and provide genome-wide characterization of memory and non-memory dehydration stress response genes in A.thaliana. The transcriptional responses during repeated exposures to stress are different from known responses occurring during a single exposure. GO analyses of encoded proteins suggested implications for the cellular/organismal protective, adaptive, and survival functions encoded by the memory genes. The results add a new dimension to our understanding of plants' responses to dehydration stress and to current models for interactions between different signaling systems when adjusting to repeated spells of water deficits.

Piglet cardiopulmonary bypass induces intestinal dysbiosis and barrier dysfunction associated with systemic inflammation.

The intestinal microbiome is essential to human health and homeostasis, and is implicated in the pathophysiology of disease, including congenital heart disease and cardiac surgery. Improving the microbiome and reducing inflammatory metabolites may reduce systemic inflammation following cardiac surgery with cardiopulmonary bypass (CPB) to expedite recovery post-operatively. Limited research exists in this area and identifying animal models that can replicate changes in the human intestinal microbiome after CPB is necessary. We used a piglet model of CPB with two groups, CPB (n=5) and a control group with mechanical ventilation (n=7), to evaluate changes to the microbiome, intestinal barrier dysfunction and intestinal metabolites with inflammation after CPB. We identified significant changes to the microbiome, barrier dysfunction, intestinal short-chain fatty acids and eicosanoids, and elevated cytokines in the CPB/deep hypothermic circulatory arrest group compared to the control group at just 4 h after intervention. This piglet model of CPB replicates known human changes to intestinal flora and metabolite profiles, and can be used to evaluate gut interventions aimed at reducing downstream inflammation after cardiac surgery with CPB.

Targeting the ATF6-Mediated ER Stress Response and Autophagy Blocks Integrin-Driven Prostate Cancer Progression.

Prostate cancer progression to the lethal metastatic castration-resistant phenotype (mCRPC) is driven by αv integrins and is associated with Golgi disorganization and activation of the ATF6 branch of unfolded protein response (UPR). Overexpression of integrins requires N-acetylglucosaminyltransferase-V (MGAT5)-mediated glycosylation and subsequent cluster formation with Galectin-3 (Gal-3). However, the mechanism underlying this altered glycosylation is missing. For the first time, using HALO analysis of IHC, we found a strong association of integrin αv and Gal-3 at the plasma membrane (PM) in primary prostate cancer and mCRPC samples. We discovered that MGAT5 activation is caused by Golgi fragmentation and mislocalization of its competitor, N-acetylglucosaminyltransferase-III, MGAT3, from Golgi to the endoplasmic reticulum (ER). This was validated in an ethanol-induced model of ER stress, where alcohol treatment in androgen-refractory PC-3 and DU145 cells or alcohol consumption in patient with prostate cancer samples aggravates Golgi scattering, activates MGAT5, and enhances integrin expression at PM. This explains known link between alcohol consumption and prostate cancer mortality. ATF6 depletion significantly blocks UPR and reduces the number of Golgi fragments in both PC-3 and DU145 cells. Inhibition of autophagy by hydroxychloroquine (HCQ) restores compact Golgi, rescues MGAT3 intra-Golgi localization, blocks glycan modification via MGAT5, and abrogates delivery of Gal-3 to the cell surface. Importantly, the loss of Gal-3 leads to reduced integrins at PM and their accelerated internalization. ATF6 depletion and HCQ treatment synergistically decrease integrin αv and Gal-3 expression and temper orthotopic tumor growth and metastasis. IMPLICATIONS: Combined ablation of ATF6 and autophagy can serve as new mCRPC therapeutic.

Microarray analysis of gene expression profiles in cells transfected with nonviral vectors.

Inefficient gene delivery is a critical factor limiting the use of nonviral methods in therapeutic applications including gene therapy and tissue engineering. There have been few efforts to understand or engineer the molecular signaling pathways that dictate the efficacy of gene transfer. Microarray analysis was used to determine endogenous gene expression profiles modulated during nonviral gene transfer. Nonviral DNA lipoplexes were delivered to HEK 293T cells. Flow cytometry was used to isolate a population of transfected cells. Expression patterns were compared between transfected and nontransfected samples, which revealed three genes that were significantly upregulated in transfected cells, including RAP1A, a GTPase implicated in integrin-mediated cell adhesion, and HSP70B', a stress-inducible gene that may be important for maintaining cell viability. Furthermore, RAP1A was also significantly upregulated in untransfected cells that were exposed to lipoplexes but that had not expressed the transgene as compared to control, untreated cells. Transfection in the presence of activators of upregulated genes was enhanced, demonstrating the principle of altering endogenous gene expression profiles to enhance transfection. With a greater understanding of signaling pathways involved in gene delivery, more efficient nonviral delivery schemes capitalizing on endogenous factors can be developed to advance therapeutic applications.

A Monovalent Mt10-CVB3 Vaccine Prevents CVB4-Accelerated Type 1 Diabetes in NOD Mice.

Enteroviruses, which include Coxsackieviruses, are a common cause of virus infections in humans, and multiple serotypes of the group B Coxsackievirus (CVB) can induce similar diseases. No vaccines are currently available to prevent CVB infections because developing serotype-specific vaccines is not practical. Thus, developing a vaccine that induces protective immune responses for multiple serotypes is desired. In that direction, we created a live-attenuated CVB3 vaccine virus, designated mutant (Mt)10, that offers protection against myocarditis and pancreatitis induced by CVB3 and CVB4 in disease-susceptible A/J mice. Here, we report that the Mt10 vaccine protected against CVB4-triggered type 1 diabetes (T1D) in non-obese diabetic (NOD) mice but the expected subsequent development of spontaneous T1D in these genetically predisposed NOD mice was not altered. We noted that Mt10 vaccine induced significant amounts of neutralizing antibodies, predominantly of the IgG2c isotype, and the virus was not detected in vaccine-challenged animals. Furthermore, monitoring blood glucose levels-and to a lesser extent, insulin antibodies-was found to be helpful in predicting vaccine responses. Taken together, our data suggest that the monovalent Mt10 vaccine has the potential to prevent infections caused by multiple CVB serotypes, as we have demonstrated in various pre-clinical models.