Bacterial-derived uracil as a modulator of mucosal immunity and gut-microbe homeostasis in Drosophila.

All metazoan guts are subjected to immunologically unique conditions in which an efficient antimicrobial system operates to eliminate pathogens while tolerating symbiotic commensal microbiota. However, the molecular mechanisms controlling this process are only partially understood. Here, we show that bacterial-derived uracil acts as a ligand for dual oxidase (DUOX)-dependent reactive oxygen species generation in Drosophila gut and that the uracil production in bacteria causes inflammation in the gut. The acute and controlled uracil-induced immune response is required for efficient elimination of bacteria, intestinal cell repair, and host survival during infection of nonresident species. Among resident gut microbiota, uracil production is absent in symbionts, allowing harmonious colonization without DUOX activation, whereas uracil release from opportunistic pathobionts provokes chronic inflammation. These results reveal that bacteria with distinct abilities to activate uracil-induced gut inflammation, in terms of intensity and duration, act as critical factors that determine homeostasis or pathogenesis in gut-microbe interactions.

Identification and characterization of GAL4 drivers that mark distinct cell types and regions in the Drosophila adult gut.

The gastrointestinal tract in the adult Drosophila serves as a model system for exploring the mechanisms underlying digestion, absorption and excretion, stem cell plasticity, and inter-organ communication, particularly through the gut-brain axis. It is also useful for studying the cellular and adaptive responses to dietary changes, alterations in microbiota and immunity, and systematic and endocrine signals. Despite the various cell types and distinct regions in the gastrointestinal tract, few tools are available to target and manipulate the activity of each cell type and region, and their gene expression. Here, we report 353 GAL4 lines and several split-GAL4 lines that are expressed in enteric neurons (ENs), progenitors (ISCs and EBs), enterocytes (ECs), enteroendocrine cells (EEs), or/and other cell types that are yet to be identified in distinct regions of the gut. We had initially collected approximately 600 GAL4 lines that may be expressed in the gut based on RNA sequencing data, and then crossed them to UAS-GFP to perform immunohistochemistry to identify those that are expressed selectively in the gut. The cell types and regional expression patterns that are associated with the entire set of GAL4 drivers and split-GAL4 combinations are annotated online at http://kdrc.kr/index.php (K-Gut Project). This GAL4 resource can be used to target specific populations of distinct cell types in the fly gut, and therefore, should permit a more precise investigation of gut cells that regulate important biological processes.

Genetic evidence of a redox-dependent systemic wound response via Hayan protease-phenoloxidase system in Drosophila.

Systemic wound response (SWR) through intertissue communication in response to local wounds is an essential biological phenomenon that occurs in all multicellular organisms from plants to animals. However, our understanding of SWR has been greatly hampered by the complexity of wound signalling communication operating within the context of an entire organism. Here, we show genetic evidence of a redox-dependent SWR from the wound site to remote tissues by identifying critical genetic determinants of SWR. Local wounds in the integument rapidly induce activation of a novel circulating haemolymph serine protease, Hayan, which in turn converts pro-phenoloxidase (PPO) to phenoloxidase (PO), an active form of melanin-forming enzyme. The Haemolymph Hayan-PO cascade is required for redox-dependent activation of the c-Jun N-terminal kinase (JNK)-dependent cytoprotective program in neuronal tissues, thereby achieving organism level of homeostasis to resist local physical trauma. These results imply that the PO-activating enzyme cascade, which is a prominent defense system in humoral innate immunity, also mediates redox-dependent SWR, providing a novel link between wound response and the nervous system.

Draft genome sequence of Gluconobacter morbifer G707T, a pathogenic gut bacterium isolated from Drosophila melanogaster intestine.

Gluconobacter morbifer G707(T), a minor member of gut microbiota, was isolated from fruit fly (Drosophila melanogaster). Here, the draft genome sequence of Gluconobacter morbifer G707(T) is reported.

Draft genome sequence of Commensalibacter intestini A911T, a symbiotic bacterium isolated from Drosophila melanogaster intestine.

Commensalibacter intestini A911(T), a predominant symbiotic bacterium capable of stably colonizing gut epithelia, was isolated from the fruit fly, Drosophila melanogaster. Here we report the draft genome sequence of Commensalibacter intestini A911(T).

Vitamin D intake and bone mineral density in Korean adults: analysis of the 2009-2011 Korea National Health and Nutrition Examination Survey.

BACKGROUND/OBJECTIVES: The prevalence of vitamin D deficiency in Koreans is quite high; however, until recently, Korean National Health and Nutrition Survey (KNHANES) had not analyzed the vitamin D intake among Koreans. Additionally, the Korean Dietary Reference Intake for vitamin D was established based on insufficient evidence. Therefore, we investigated vitamin D intake and its relationship with bone mineral density (BMD) in Korean adults using the combined data from the 2009-2011 KNHANES. MATERIALS AND METHODS: This study was conducted in 11,949 healthy adults. Vitamin D intake was assessed using a 24-h recall method, and the BMD was measured using dual-energy X-ray absorptiometry. RESULTS: The prevalence of vitamin D deficiency (< 20 ng/mL) was 64% in men and 77% in women. In women aged ≥ 50 yrs and men aged < 50 yrs, there was a significant positive correlation between vitamin D intake and serum 25-hydroxyvitamin D level after sun exposure adjustment. The BMD of postmenopausal women aged ≥ 50 yrs with a vitamin D intake of 5 µg/day or more was significantly higher than that of women with intake less than 5 µg/day. After adjusting for age, energy, and calcium intake, the vitamin D intake of the osteoporotic group was significantly lower than that of the osteopenia group in women. CONCLUSIONS: Since the relationship between vitamin D intake and BMD was observed in women aged ≥ 50 yrs, further research is needed to clarify these findings using cohort or randomized controlled trials.

Heat shock protein 60 couples an oxidative stress-responsive p38/MK2 signaling and NF-κB survival machinery in cancer cells.

Mitochondria communicate with other cellular compartments via the secretion of protein factors. Here, we report an unexpected messenger role for heat shock protein 60 (HSP60) as a mitochondrial-releasing protein factor that couples stress-sensing signaling and cell survival machineries. We show that mild oxidative stress predominantly activates the p38/MK2 complex, which phosphorylates mitochondrial fission factor 1 (MFF1) at the S155 site. Such phosphorylated MFF1 leads to the oligomerization of voltage anion-selective channel 1, thereby triggering the formation of a mitochondrial membrane pore through which the matrix protein HSP60 passes. The liberated HSP60 associates with and activates the IκB kinase (IKK) complex in the cytosol, which consequently induces the NF-κB-dependent expression of survival genes in nucleus. Indeed, inhibition of the HSP60 release or HSP60-IKK interaction sensitizes the cancer cells to mild oxidative stress and regresses the tumorigenic growth of cancer cells in the mouse xenograft model. Thus, this study reveals a novel mitonuclear survival axis responding to oxidative stress.

Characterization of Proteins Regulated by Androgen and Protein Kinase a Signaling in VCaP Prostate Cancer Cells.

Androgen signaling via the androgen receptor (AR) is involved in normal prostate development and prostate cancer progression. In addition to androgen binding, a variety of protein kinases, including cyclic AMP-dependent protein kinase A (PKA), can activate the AR. Although hormone deprivation, especially that of androgen, continues to be an important strategy for treating prostate cancer patients, the disease ultimately progresses to castration-resistant prostate cancer (CRPC), despite a continuous hormone-deprived environment. To date, it remains unclear which pathways in this progression are active and targetable. Here, we performed a proteomic analysis of VCaP cells stimulated with androgen or forskolin to identify proteins specific for androgen-induced and androgen-bypassing signaling, respectively. Patterns of differentially expressed proteins were quantified, and eight proteins showing significant changes in expression were identified. Functional information, including a Gene Ontology analysis, revealed that most of these proteins are involved in metabolic processes and are associated with cancer. The mRNA and protein expression of selected proteins was validated, and functional correlations of identified proteins with signaling in VCaP cells were assessed by measuring metabolites related to each enzyme. These analyses offered new clues regarding effector molecules involved in prostate cancer development, insights that are supported by the demonstration of increased expression levels of the eight identified proteins in prostate cancer patients and assessments of the progression-free interval. Taken together, our findings show that aberrant levels of eight proteins reflect molecular changes that are significantly regulated by androgen and/or PKA signaling pathways, suggesting possible molecular mechanisms of CRPC.

Rapid Androgen-Responsive Proteome Is Involved in Prostate Cancer Progression.

Androgen exerts its functions by binding with an androgen receptor (AR). It can activate many signaling pathways that are important to the progression of castration-resistant prostate cancer (CRPC). Here, we characterized the rapid proteomic changes seen at 5, 15, 30, and 60 min after the androgen treatment of VCaP cells via the tandem mass tag (TMT) labeling strategy. A total of 5529 proteins were successfully identified and quantified. Dynamic time profiling of protein expression patterns allowed us to identify five protein clusters involved in various stages of androgen-initiated signal transmission and processing. More details of protein functions and localization patterns, and our elucidation of an AR-interacting protein network, were obtained. Finally, we validated the expression level of AR-regulated proteins known to be significantly regulated in CRPC patients using the mouse xenograft model and patient samples. Our work offers a systematic analysis of the rapid proteomic changes induced by androgen and provides a global view of the molecular mechanisms underlying CRPC progression.

Prediction of IDH1 Mutation Status in Glioblastoma Using Machine Learning Technique Based on Quantitative Radiomic Data.

OBJECTIVE: Isocitrate dehydrogenase 1 (IDH1) mutation status is an independent favorable prognostic factor for glioblastoma (GBM) and is usually determined by sequencing or immunohistochemistry. An accurate prediction of IDH1 mutation status via noninvasive methods helps establish the appropriate treatment strategy. We aimed to predict IDH1 mutation status using quantitative radiomic data in patients with GBM. METHODS: Between May 2010 and June 2015, we retrospectively identified 88 patients with newly diagnosed GBM. After semiautomatic segmentation of the lesions, we extracted 31 features from preoperative multiparametric magnetic resonance images. We also determined IDH1 mutation status using targeted sequencing and immunohistochemistry. A training cohort (n = 88) was used to train machine learning-based classifiers, with internal validation. The machine-learning technique was then validated in an external dataset of 35 patients with GBM. RESULTS: We detected the IDH1 mutation in 12 out of 88 GBMs. Multiparametric radiomic profiles revealed that the IDH1 mutation was associated with a smaller enhancing area volume and a larger necrotic area volume. Using the machine learning-based classification algorithms, we identified 70.3%-87.3% of prediction rate of IDH1 mutation status and found 66.3%-83.4% accuracy in the external validation set. CONCLUSIONS: We demonstrate that machine learning algorithms can predict IDH1 mutation status in GBM using preoperative multiparametric magnetic resonance images.

Tumor necrosis factor-alpha develops late anaphylactic reaction through cytosolic phospholipase A(2) activation.

BACKGROUND: We have recently reported that tumor necrosis factor (TNF)-alpha plays an important role in the development of a late anaphylactic reaction, but the downstream pathway beyond TNF-alpha remains unclear. OBJECTIVE: It was the aim of this study to examine whether TNF-alpha induces late-phase anaphylaxis via the activation of cytosolic phospholipase A(2) (cPLA(2)). METHODS: Using a murine model of active systemic anaphylaxis to penicillin V, the induction of the late phase of anaphylaxis was quantified by measuring the increase in hematocrit value as well as the plasma level of platelet-activating factor in TNF-alpha knockout mice. Phosphorylation of mitogen-activated protein kinases (MAPKs) and cPLA(2) was measured by immunoprecipitation. cPLA(2) activity was assessed by using 1-stearoyl-2-[1-(14)C] arachidonyl-sn-glycero-3-phosphocholine as the substrate. RESULTS: Phosphorylation and enzymatic activity of cPLA(2), and phosphorylation of the 3 known MAPKs, i.e. p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase, were markedly increased in a TNF-alpha-dependent way in the lungs of mice undergoing anaphylaxis. A specific cPLA(2) inhibitor significantly attenuated the late anaphylactic symptoms. Either p38 or an ERK inhibitor significantly attenuated not only cPLA(2) phosphorylation and activity, but also the late-phase anaphylaxis. CONCLUSION: TNF-alpha-induces cPLA(2) activation through the pathway involving p38 MAPK and ERK activation and appears to be the key mechanism leading to the development of late-phase anaphylaxis.

Quantitative radiomic profiling of glioblastoma represents transcriptomic expression.

Quantitative imaging biomarkers have increasingly emerged in the field of research utilizing available imaging modalities. We aimed to identify good surrogate radiomic features that can represent genetic changes of tumors, thereby establishing noninvasive means for predicting treatment outcome. From May 2012 to June 2014, we retrospectively identified 65 patients with treatment-naïve glioblastoma with available clinical information from the Samsung Medical Center data registry. Preoperative MR imaging data were obtained for all 65 patients with primary glioblastoma. A total of 82 imaging features including first-order statistics, volume, and size features, were semi-automatically extracted from structural and physiologic images such as apparent diffusion coefficient and perfusion images. Using commercially available software, NordicICE, we performed quantitative imaging analysis and collected the dataset composed of radiophenotypic parameters. Unsupervised clustering methods revealed that the radiophenotypic dataset was composed of three clusters. Each cluster represented a distinct molecular classification of glioblastoma; classical type, proneural and neural types, and mesenchymal type. These clusters also reflected differential clinical outcomes. We found that extracted imaging signatures does not represent copy number variation and somatic mutation. Quantitative radiomic features provide a potential evidence to predict molecular phenotype and treatment outcome. Radiomic profiles represents transcriptomic phenotypes more well.

Glutamine inhibits lipopolysaccharide-induced cytoplasmic phospholipase A2 activation and protects against endotoxin shock in mouse.

Glutamine (Gln) supplementation is known to play a beneficial role in a number of settings of critical illness as well as laboratory models of endotoxin shock. We have investigated a molecular mechanism of the protective role of Gln in lipopolysaccharide (LPS)-induced shock using a mouse model. To examine the effectiveness of Gln, Gln was administered before or after LPS injection. Treatment of Gln before, but not after, LPS injection resulted in inhibition of nuclear factor kappaB activation and tumor necrosis factor alpha synthesis. In contrast, protection of animal from LPS-mediated death by Gln was observed when the Gln treatment was performed after LPS injection, suggesting that nuclear factor kappaB/tumor necrosis factor alpha signaling does not play an important role in this process. LPS injection induced phosphorylation of cytoplasmic phospholipase A2 (cPLA2), which was blocked by Gln treatment after LPS injection. Similarly, the LPS-stimulated cPLA2 activity was also inhibited by Gln treatment after LPS injection. Moreover, a cPLA2 inhibitor not only inhibited LPS-induced activation of cPLA2, but also significantly prevented LPS-mediated death. These observations indicate that Gln has a capability to inhibit cPLA2 phosphorylation and activation and suggest that Gln might be of a great therapeutic value for controlling inflammatory diseases in which cPLA2 plays an important role in the pathogenesis of the diseases.

Uracil-induced signaling pathways for DUOX-dependent gut immunity.

Intestinal dual oxidase (DUOX) activation is the first line of host defense against enteric infection in Drosophila. DUOX enzymatic activity is mainly controlled by phospholipase C-ß (PLCß)-dependent calcium mobilization, whereas DUOX gene expression is mainly controlled by the MEKK1-p38 mitogen-activated protein kinase pathway. Furthermore, bacterial-derived uracil molecules act as ligands for DUOX activation. However, our current understanding of uracil-induced signal transduction pathways remain incomplete. We have recently found that uracil stimulates Hedgehog signaling, which in turn upregulates cadherin99C (Cad99C) expression in enterocytes. Cad99C molecules, along with PLCß and protein kinase C, induce the formation of signaling endosomes that facilitate intracellular calcium mobilization for DUOX activity. These observations illustrate the complexity of signaling cascades in uracil-induced signaling pathways. Here, we further demonstrated the role of lipid raft formation and calmodulin-dependent protein kinase-II on endosome formation and calcium mobilization, respectively. Moreover, we will provide a brief discussion on two different models for uracil recognition and uracil-induced DUOX activation in Drosophila enterocytes.

Bacterial uracil modulates Drosophila DUOX-dependent gut immunity via Hedgehog-induced signaling endosomes.

Genetic studies in Drosophila have demonstrated that generation of microbicidal reactive oxygen species (ROS) through the NADPH dual oxidase (DUOX) is a first line of defense in the gut epithelia. Bacterial uracil acts as DUOX-activating ligand through poorly understood mechanisms. Here, we show that the Hedgehog (Hh) signaling pathway modulates uracil-induced DUOX activation. Uracil-induced Hh signaling is required for intestinal expression of the calcium-dependent cell adhesion molecule Cadherin 99C (Cad99C) and subsequent Cad99C-dependent formation of endosomes. These endosomes play essential roles in uracil-induced ROS production by acting as signaling platforms for PLCß/PKC/Ca2+-dependent DUOX activation. Animals with impaired Hh signaling exhibit abolished Cad99C-dependent endosome formation and reduced DUOX activity, resulting in high mortality during enteric infection. Importantly, endosome formation, DUOX activation, and normal host survival are restored by genetic reintroduction of Cad99C into enterocytes, demonstrating the important role for Hh signaling in host resistance to enteric infection.

Homeostasis between gut-associated microorganisms and the immune system in Drosophila.

The metabolic activities of a given gut bacterium or gut commensal community fluctuate in a manner largely depending on the physicochemical parameters within the gut niche. Recognition of the bacterial metabolic status in situ, by a sensing of the gut metabolites as a signature of a specific bacterial metabolic activity, has been suggested to be a highly beneficial means for the host to maintain gut-microbe homeostasis. Recently, analysis of Drosophila gut immunity revealed that bacterial-derived uracil and uracil-modulated intestinal reactive oxygen species (ROS) generation play a pivotal role in diverse aspects of host-microbe interactions, such as pathogen clearance, commensal protection, intestinal cell regeneration, colitogenesis, and possibly also interorgan immunological communication. A deeper understanding of the role of uracil in Drosophila immunity will provide additional insight into the molecular mechanisms underlying host-microbe symbiosis and dysbiosis.

Drosophila microbiome modulates host developmental and metabolic homeostasis via insulin signaling.

The symbiotic microbiota profoundly affect many aspects of host physiology; however, the molecular mechanisms underlying host-microbe cross-talk are largely unknown. Here, we show that the pyrroloquinoline quinone-dependent alcohol dehydrogenase (PQQ-ADH) activity of a commensal bacterium, Acetobacter pomorum, modulates insulin/insulin-like growth factor signaling (IIS) in Drosophila to regulate host homeostatic programs controlling developmental rate, body size, energy metabolism, and intestinal stem cell activity. Germ-free animals monoassociated with PQQ-ADH mutant bacteria displayed severe deregulation of developmental and metabolic homeostasis. Importantly, these defects were reversed by enhancing host IIS or by supplementing the diet with acetic acid, the metabolic product of PQQ-ADH.

Endocytosis of the type III transforming growth factor-beta (TGF-beta) receptor through the clathrin-independent/lipid raft pathway regulates TGF-beta signaling and receptor down-regulation.

Transforming growth factor-beta (TGF-beta) signals through three highly conserved cell surface receptors, the type III TGF-beta receptor (T beta RIII), the type II TGF-beta receptor (T beta RII), and the type I TGF-beta receptor (T beta RI) to regulate diverse cellular processes including cell proliferation, differentiation, migration, and apoptosis. Although T beta RI and T beta RII undergo ligand-independent endocytosis by both clathrin-mediated endocytosis, resulting in enhanced signaling, and clathrin-independent endocytosis, resulting in receptor degradation, the mechanism and function of T beta RIII endocytosis is poorly understood. T beta RIII is a heparan sulfate proteoglycan with a short cytoplasmic tail that functions as a TGF-beta superfamily co-receptor, contributing to TGF-beta signaling through mechanisms yet to be fully defined. We have reported previously that T beta RIII endocytosis, mediated by a novel interaction with beta arrestin-2, results in decreased TGF-beta signaling. Here we demonstrate that T beta RIII undergoes endocytosis in a ligand and glycosaminoglycan modification-independent and cytoplasmic domain-dependent manner, with the interaction of Thr-841 in the cytoplasmic domain of T beta RIII with beta-arrestin2 enhancing T beta RIII endocytosis. T beta RIII undergoes both clathrin-mediated and clathrin-independent endocytosis. Importantly, inhibition of the clathrin-independent, lipid raft pathway, but not of the clathrin-dependent pathway, results in decreased TGF-beta1 induced Smad2 and p38 phosphorylation, supporting a specific role for clathrin-independent endocytosis of T beta RIII in regulating both Smad-dependent and Smad-independent TGF-beta signaling.

Inhibition of receptor internalization attenuates the TNFalpha-induced ROS generation in non-phagocytic cells.

Reactive oxygen species (ROS) are important regulatory molecules implicated in the signaling cascade triggered by tumor necrosis factor (TNF)alpha, although the events through which TNFalpha induces ROS generation are not well characterized. Here, we report that TNFalpha-induced ROS production was blocked by pretreatment with internalization inhibitor monodansyl cadaverine (MDC). Similarly, a transient expression of a GTP-binding and hydrolysis-defective dynamin mutant (dynamin(K44A)) that had been shown to be defective in internalization significantly attenuated the TNFalpha-induced intracellular ROS production. Importantly, the inhibition of receptor internalization suppressed TNFalpha signaling to mitogen-activated protein kinases (MAPKs) stimulation. Together, our results suggest that receptor internalization is somehow necessary for the TNFalpha-induced ROS generation and subsequent intracellular downstream signaling in non-phagocytes.

Nordihydroguaiaretic acid inhibits IFN-gamma-induced STAT tyrosine phosphorylation in rat brain astrocytes.

The Janus kinase (JAK) and signal transducers and activators of transcription (STAT) signal cascades are major pathways that mediate the inflammatory functions of interferon-gamma (IFN-gamma), an important pro-inflammatory cytokine. Therefore, regulation of JAK/STAT signaling should modulate IFN-gamma-mediated inflammation. In this study, we found that nordihydroguaiaretic acid (NDGA), a well-known lipoxygenase (LO) inhibitor, suppressed IFN-gamma-induced inflammatory responses in brain astrocytes. In the presence of NDGA, interferon regulatory factor-1 expression was significantly reduced. Expression of monocyte chemotactic protein-1 and interferon-gamma inducible protein-10 mRNA in response to IFN-gamma was significantly suppressed in the presence of NDGA, as was tyrosine-phosphorylation of JAK and STAT. However, the 5-LO products, leukotriene B(4) (LTB(4)) and leukotriene C(4), were not detected in cells treated with IFN-gamma, indicating that the effect of NDGA seemed to be independent of 5-LO inhibition. In addition, two other 5-LO inhibitors (Rev5901 and AA861) did not mimic the effect of NDGA, and the 5-LO metabolites, 5-hydroxyeicosatetraenoic acid and LTB(4), were unable to reverse NDGA-driven suppression of STAT activation or affect basal STAT phosphorylation. Taken together, these results suggest that NDGA regulates IFN-gamma-mediated inflammation through mechanisms unrelated to the inhibition of 5-LO.