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.

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.

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).

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.

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.

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.

Genome structure and evolutionary history of frankincense producing Boswellia sacra.

Boswellia sacra Flueck (family Burseraceae) tree is wounded to produce frankincense. We report its de novo assembled genome (667.8 Mb) comprising 18,564 high-confidence protein-encoding genes. Comparing conserved single-copy genes across eudicots suggest >97% gene space assembly of B. sacra genome. Evolutionary history shows B. sacra gene-duplications derived from recent paralogous events and retained from ancient hexaploidy shared with other eudicots. The genome indicated a major expansion of Gypsy retroelements in last 2 million years. The B. sacra genetic diversity showed four clades intermixed with a primary genotype-dominating most resin-productive trees. Further, the stem transcriptome revealed that wounding concurrently activates phytohormones signaling, cell wall fortification, and resin terpenoid biosynthesis pathways leading to the synthesis of boswellic acid-a key chemotaxonomic marker of Boswellia. The sequence datasets reported here will serve as a foundation to investigate the genetic determinants of frankincense and other resin-producing species in Burseraceae.

The non-canonical mechanism of ER stress-mediated progression of prostate cancer.

BACKGROUND: The development of persistent endoplasmic reticulum (ER) stress is one of the cornerstones of prostate carcinogenesis; however, the mechanism is missing. Also, alcohol is a physiological ER stress inducer, and the link between alcoholism and progression of prostate cancer (PCa) is well documented but not well characterized. According to the canonical model, the mediator of ER stress, ATF6, is cleaved sequentially in the Golgi by S1P and S2P proteases; thereafter, the genes responsible for unfolded protein response (UPR) undergo transactivation. METHODS: Cell lines used were non-malignant prostate epithelial RWPE-1 cells, androgen-responsive LNCaP, and 22RV1 cells, as well as androgen-refractory PC-3 cells. We also utilized PCa tissue sections from patients with different Gleason scores and alcohol consumption backgrounds. Several sophisticated approaches were employed, including Structured illumination superresolution microscopy, Proximity ligation assay, Atomic force microscopy, and Nuclear magnetic resonance spectroscopy. RESULTS: Herein, we identified the trans-Golgi matrix dimeric protein GCC185 as a Golgi retention partner for both S1P and S2P, and in cells lacking GCC185, these enzymes lose intra-Golgi situation. Progression of prostate cancer (PCa) is associated with overproduction of S1P and S2P but monomerization of GCC185 and its downregulation. Utilizing different ER stress models, including ethanol administration, we found that PCa cells employ an elegant mechanism that auto-activates ER stress by fragmentation of Golgi, translocation of S1P and S2P from Golgi to ER, followed by intra-ER cleavage of ATF6, accelerated UPR, and cell proliferation. The segregation of S1P and S2P from Golgi and activation of ATF6 are positively correlated with androgen receptor signaling, different disease stages, and alcohol consumption. Finally, depletion of ATF6 significantly retarded the growth of xenograft prostate tumors and blocks production of pro-metastatic metabolites. CONCLUSIONS: We found that progression of PCa associates with translocation of S1P and S2P proteases to the ER and subsequent ATF6 cleavage. This obviates the need for ATF6 transport to the Golgi and enhances UPR and cell proliferation. Thus, we provide the novel mechanistic model of ATF6 activation and ER stress implication in the progression of PCa, suggesting ATF6 is a novel promising target for prostate cancer therapy.

Exon-primed intron-crossing (EPIC) markers for non-model teleost fishes.

BACKGROUND: Exon-primed intron-crossing (EPIC) markers have three advantages over anonymous genomic sequences in studying evolution of natural populations. First, the universal primers designed in exon regions can be applied across a broad taxonomic range. Second, the homology of EPIC-amplified sequences can be easily determined by comparing either their exon or intron portion depending on the genetic distance between the taxa. Third, having both the exon and intron fragments could help in examining genetic variation at the intraspecific and interspecific level simultaneously, particularly helpful when studying species complex. However, the paucity of EPIC markers has hindered multilocus studies using nuclear gene sequences, particularly in teleost fishes. RESULTS: We introduce a bioinformatics pipeline for developing EPIC markers by comparing the whole genome sequences between two or more species. By applying this approach on five teleost fishes whose genomes were available in the Ensembl database http://www.ensembl.org, we identified 210 EPIC markers that have single-copy and conserved exon regions with identity greater than 85% among the five teleost fishes. We tested 12 randomly chosen EPIC markers in nine teleost species having a wide phylogenetic range. The success rate of amplifying and sequencing those markers varied from 44% to 100% in different species. We analyzed the exon sequences of the 12 EPIC markers from 13 teleosts. The resulting phylogeny contains many traditionally well-supported clades, indicating the usefulness of the exon portion of EPIC markers in reconstructing species phylogeny, in addition to the value of the intron portion of EPIC markers in interrogating the population history. CONCLUSIONS: This study illustrated an effective approach to develop EPIC markers in a taxonomic group, where two or more genome sequences are available. The markers identified could be amplified across a broad taxonomic range of teleost fishes. The phylogenetic utility of individual markers varied according to intron size and amplifiability. The bioinformatics pipelines developed are readily adapted to other taxonomic groups.

Dynamic changes in genome-wide histone H3 lysine 4 methylation patterns in response to dehydration stress in Arabidopsis thaliana.

BACKGROUND: The molecular mechanisms of genome reprogramming during transcriptional responses to stress are associated with specific chromatin modifications. Available data, however, describe histone modifications only at individual plant genes induced by stress. We have no knowledge of chromatin modifications taking place at genes whose transcription has been down-regulated or on the genome-wide chromatin modification patterns that occur during the plant's response to dehydration stress. RESULTS: Using chromatin immunoprecipitation and deep sequencing (ChIP-Seq) we established the whole-genome distribution patterns of histone H3 lysine 4 mono-, di-, and tri-methylation (H3K4me1, H3K4me2, and H3K4me3, respectively) in Arabidopsis thaliana during watered and dehydration stress conditions. In contrast to the relatively even distribution of H3 throughout the genome, the H3K4me1, H3K4me2, and H3K4me3 marks are predominantly located on genes. About 90% of annotated genes carry one or more of the H3K4 methylation marks. The H3K4me1 and H3K4me2 marks are more widely distributed (80% and 84%, respectively) than the H3K4me3 marks (62%), but the H3K4me2 and H3K4me1 levels changed only modestly during dehydration stress. By contrast, the H3K4me3 abundance changed robustly when transcripts levels from responding genes increased or decreased. In contrast to the prominent H3K4me3 peaks present at the 5'-ends of most transcribed genes, genes inducible by dehydration and ABA displayed atypically broader H3K4me3 distribution profiles that were present before and after the stress. CONCLUSIONS: A higher number (90%) of annotated Arabidopsis genes carry one or more types of H3K4me marks than previously reported. During the response to dehydration stress the changes in H3K4me1, H3K4me2, and H3K4me3 patterns show different dynamics and specific patterns at up-regulated, down-regulated, and unaffected genes. The different behavior of each methylation mark during the response process illustrates that they have distinct roles in the transcriptional response of implicated genes. The broad H3K4me3 distribution profiles on nucleosomes of stress-induced genes uncovered a specific chromatin pattern associated with many of the genes involved in the dehydration stress response.

Host Transcriptional Response to Persistent Infection with a Live-Attenuated Porcine Reproductive and Respiratory Syndrome Virus Strain.

Both virulent and live-attenuated porcine reproductive and respiratory syndrome virus (PRRSV) strains can establish persistent infection in lymphoid tissues of pigs. To investigate the mechanisms of PRRSV persistence, we performed a transcriptional analysis of inguinal lymphoid tissue collected from pigs experimentally infected with an attenuated PRRSV strain at 46 days post infection. A total of 6404 differentially expressed genes (DEGs) were detected of which 3960 DEGs were upregulated and 2444 DEGs were downregulated. Specifically, genes involved in innate immune responses and chemokines and receptors associated with T-cell homing to lymphoid tissues were down regulated. As a result, homing of virus-specific T-cells to lymphoid tissues seems to be ineffective, evidenced by the lower frequencies of virus-specific T-cell in lymphoid tissue than in peripheral blood. Genes associated with T-cell exhaustion were upregulated. Likewise, genes involved in the anti-apoptotic pathway were upregulated. Collectively, the data suggested that the live-attenuated PRRSV strain establishes a pro-survival microenvironment in lymphoid tissue by suppressing innate immune responses, T-cell homing, and preventing cell apoptosis.

The Role of Alcohol-Induced Golgi Fragmentation for Androgen Receptor Signaling in Prostate Cancer.

Multiple epidemiologic observations and meta-analysis clearly indicate the link between alcohol abuse and the incidence and progression of prostate cancer; however, the mechanism remains enigmatic. Recently, it was found that ethanol (EtOH) induces disorganization of the Golgi complex caused by impaired function of the largest Golgi matrix protein, giantin (GOLGB1), which, in turn, alters the Golgi docking of resident Golgi proteins. Here, it is determined that in normal prostate cells, histone deacetylase 6 (HDAC6), the known regulator of androgen receptor (AR) signaling, localizes in the cytoplasm and nucleus, while its kinase, glycogen synthase kinase ß (GSK3ß), primarily resides in the Golgi. Progression of prostate cancer is accompanied by Golgi scattering, translocation of GSK3ß from the Golgi to the cytoplasm, and the cytoplasmic shift in HDAC6 localization. Alcohol dehydrogenase-generated metabolites induces Golgi disorganization in androgen-responsive LNCaP and 22Rv1 cells, facilitates tumor growth in a mouse xenograft model and activates anchorage-independent proliferation, migration, and cell adhesion. EtOH-treated cells demonstrate reduced giantin and subsequent cytoplasmic GSK3ß; this phenomenon was validated in giantin-depleted cells. Redistribution of GSK3ß to the cytoplasm results in phosphorylation of HDAC6 and its retention in the cytoplasm, which, in turn, stimulates deacetylation of HSP90, AR import into the nucleus, and secretion of prostate-specific antigen (PSA). Finally, the relationship between Golgi morphology, HDAC6 cytoplasmic content, and clinicopathologic features was assessed in human prostate cancer patient specimens with and without a history of alcohol dependence. IMPLICATIONS: This study demonstrates the importance of alcohol-induced Golgi fragmentation in the activation of AR-mediated proliferation.

Post-ER Stress Biogenesis of Golgi Is Governed by Giantin.

BACKGROUND: The Golgi apparatus undergoes disorganization in response to stress, but it is able to restore compact and perinuclear structure under recovery. This self-organization mechanism is significant for cellular homeostasis, but remains mostly elusive, as does the role of giantin, the largest Golgi matrix dimeric protein. METHODS: In HeLa and different prostate cancer cells, we used the model of cellular stress induced by Brefeldin A (BFA). The conformational structure of giantin was assessed by proximity ligation assay and atomic force microscopy. The post-BFA distribution of Golgi resident enzymes was examined by 3D SIM high-resolution microscopy. RESULTS: We detected that giantin is rather flexible than an extended coiled-coil dimer and BFA-induced Golgi disassembly was associated with giantin monomerization. A fusion of the nascent Golgi membranes after BFA washout is forced by giantin re-dimerization via disulfide bond in its luminal domain and assisted by Rab6a GTPase. GM130-GRASP65-dependent enzymes are able to reach the nascent Golgi membranes, while giantin-sensitive enzymes appeared at the Golgi after its complete recovery via direct interaction of their cytoplasmic tail with N-terminus of giantin. CONCLUSION: Post-stress recovery of Golgi is conducted by giantin dimer and Golgi proteins refill membranes according to their docking affiliation rather than their intra-Golgi location.

Trans-10, cis-12 conjugated linoleic acid activates the integrated stress response pathway in adipocytes.

Trans-10, cis-12 conjugated linoleic acid (t10c12 CLA) causes fat loss in mouse white adipose tissue (WAT) and adipocytes in culture. The early transcriptome changes in treated WAT and 3T3-L1 adipocytes were analyzed using high-density microarrays to better characterize the signaling pathways responding to t10c12 CLA. Gene expression responses between 4 and 24 h after treatment showed a common set of early gene expression changes indicative of an integrated stress response (ISR). The responses of 3T3-L1 preadipocytes treated with t10c12 CLA or adipocytes treated with the cis-9, trans-11 isomer of CLA did not show the ISR, indicating the effect is specific to adipocytes responding to t10c12 CLA. Western blot analysis found increased phosphorylation of eIF2 alpha and increased production of ATF4 confirming at least part of the response to t10c12 CLA is mediated through the ISR pathway. Immunofluorescence microscopy found that the cell type expressing ATF3, an indicator of the ISR, was early stage adipocytes containing oil droplets but lacking the abundant levels of fatty acid binding protein-4 (FABP4) (AP2) found in mature adipocytes. Our data suggests that the ISR precedes and is possibly the cause of the later induction of proinflammatory cytokines observed in t10c12 CLA treated adipocytes. The release of proinflammatory cytokines may explain how the ISR in early stage adipocytes causes lipid loss in mature adipocytes.

Arabidopsis MSH1 mutation alters the epigenome and produces heritable changes in plant growth.

Plant phenotypes respond to environmental change, an adaptive capacity that is at least partly transgenerational. However, epigenetic components of this interplay are difficult to measure. Depletion of the nuclear-encoded protein MSH1 causes dramatic and heritable changes in plant development, and here we show that crossing these altered plants with isogenic wild type produces epi-lines with heritable, enhanced growth vigour. Pericentromeric DNA hypermethylation occurs in a subset of msh1 mutants, indicative of heightened transposon repression, while enhanced growth epi-lines show large chromosomal segments of differential CG methylation, reflecting genome-wide reprogramming. When seedlings are treated with 5-azacytidine, root growth of epi-lines is restored to wild-type levels, implicating hypermethylation in enhanced growth. Grafts of wild-type floral stems to mutant rosettes produce progeny with enhanced growth and altered CG methylation strikingly similar to epi-lines, indicating a mobile signal when MSH1 is downregulated, and confirming the programmed nature of methylome and phenotype changes.

Dynamics of small RNA profiles of virus and host origin in wheat cultivars synergistically infected by Wheat streak mosaic virus and Triticum mosaic virus: virus infection caused a drastic shift in the endogenous small RNA profile.

Co-infection of wheat (Triticum aestivum L.) by Wheat streak mosaic virus (WSMV, a Tritimovirus) and Triticum mosaic virus (TriMV, a Poacevirus) of the family Potyviridae causes synergistic interaction. In this study, the effects of the synergistic interaction between WSMV and TriMV on endogenous and virus-derived small interfering RNAs (vsiRNAs) were examined in susceptible ('Arapahoe') and temperature-sensitive resistant ('Mace') wheat cultivars at 18°C and 27°C. Single and double infections in wheat caused a shift in the profile of endogenous small RNAs from 24 nt being the most predominant in healthy plants to 21 nt in infected wheat. Massive amounts of 21 and 22 nt vsiRNAs accumulated in singly and doubly infected Arapahoe at both temperatures and in Mace at 27°C but not 18°C. The plus- and minus-sense vsiRNAs were distributed throughout the genomic RNAs in Arapahoe at both temperature regimens and in Mace at 27°C, although some regions served as hot-spots, spawning an excessive number of vsiRNAs. The vsiRNA peaks were conserved among cultivars, suggesting that the Dicer-like enzymes in susceptible and resistant cultivars similarly accessed the genomic RNAs of WSMV or TriMV. Accumulation of large amounts of vsiRNAs in doubly infected plants suggests that the silencing suppressor proteins encoded by TriMV and WSMV do not prevent the formation of vsiRNAs; thus, the synergistic effect observed is independent from RNA-silencing mediated vsiRNA biogenesis. The high-resolution map of endogenous and vsiRNAs from WSMV- and/or TriMV-infected wheat cultivars may form a foundation for understanding the virus-host interactions, the effect of synergistic interactions on host defense, and virus resistance mechanisms in wheat.

De novo transcriptome assembly from fat body and flight muscles transcripts to identify morph-specific gene expression profiles in Gryllus firmus.

Wing polymorphism is a powerful model for examining many aspects of adaptation. The wing dimorphic cricket species, Gryllus firmus, consists of a long-winged morph with functional flight muscles that is capable of flight, and two flightless morphs. One (obligately) flightless morph emerges as an adult with vestigial wings and vestigial flight muscles. The other (plastic) flightless morph emerges with fully-developed wings but later in adulthood histolyzes its flight muscles. Importantly both flightless morphs have substantially increased reproductive output relative to the flight-capable morph. Much is known about the physiological and biochemical differences between the morphs with respect to adaptations for flight versus reproduction. In contrast, little is known about the molecular genetic basis of these morph-specific adaptations. To address this issue, we assembled a de novo transcriptome of G. firmus using 141.5 million Illumina reads generated from flight muscles and fat body, two organs that play key roles in flight and reproduction. We used the resulting 34,411 transcripts as a reference transcriptome for differential gene expression analyses. A comparison of gene expression profiles from functional flight muscles in the flight-capable morph versus histolyzed flight muscles in the plastic flight incapable morph identified a suite of genes involved in respiration that were highly expressed in pink (functional) flight muscles and genes involved in proteolysis highly expressed in the white (histolyzed) flight muscles. A comparison of fat body transcripts from the obligately flightless versus the flight-capable morphs revealed differential expression of genes involved in triglyceride biosynthesis, lipid transport, immune function and reproduction. These data provide a valuable resource for future molecular genetics research in this and related species and provide insight on the role of gene expression in morph-specific adaptations for flight versus reproduction.

EvolMarkers: a database for mining exon and intron markers for evolution, ecology and conservation studies.

Recent innovations in next-generation sequencing have lowered the cost of genome projects. Nevertheless, sequencing entire genomes for all representatives in a study remains expensive and unnecessary for most studies in ecology, evolution and conservation. It is still more cost-effective and efficient to target and sequence single-copy nuclear gene markers for such studies. Many tools have been developed for identifying nuclear markers, but most of these have focused on particular taxonomic groups. We have built a searchable database, EvolMarkers, for developing single-copy coding sequence (CDS) and exon-primed-intron-crossing (EPIC) markers that is designed to work across a broad range of phylogenetic divergences. The database is made up of single-copy CDS derived from BLAST searches of a variety of metazoan genomes. Users can search the database for different types of markers (CDS or EPIC) that are common to different sets of input species with different divergence characteristics. EvolMarkers can be applied to any taxonomic group for which genome data are available for two or more species. We included 82 genomes in the first version of EvolMarkers and have found the methods to be effective across Placozoa, Cnidaria, Arthropod, Nematoda, Annelida, Mollusca, Echinodermata, Hemichordata, Chordata and plants. We demonstrate the effectiveness of searching for CDS markers within annelids and show how to find potentially useful intronic markers within the lizard Anolis.