Two distinct pathways can control expression of the gene encoding the Drosophila antimicrobial peptide metchnikowin

Metchnikowin is a recently discovered proline-rich peptide from Drosophila with antibacterial and antifungal properties. Like most other antimicrobial peptides from insects, its expression is immune-inducible. Here we present evidence that induction of metchnikowin gene expression can be mediated either by the TOLL pathway or by the imd gene product. We show that the gene remains inducible in Toll-deficient mutants, in which the antifungal response is blocked, as well as in imd mutants, which fail to mount an antibacterial response. However, in Toll-deficient;imd double mutants, metchnikowin gene expression can no longer be detected after immune challenge. Our results suggest that expression of this peptide with dual activity can be triggered by signals generated by either bacterial or fungal infection. Cloning of the metchnikowin gene revealed the presence in the 5' flanking region of several putative cis-regulatory motifs characterized in the promoters of insect immune genes: namely, Rel sites, GATA motifs, interferon consensus response elements and NF-IL6 response elements. Establishment of transgenic fly lines in which the GFP reporter gene was placed under the control of 1.5 kb of metchnikowin gene upstream sequences indicates that this fragment is able to confer full immune inducibility and tissue specificity of expression on the transgene

Lower provider volume is associated with higher failure rates for endoscopic retrograde cholangiopancreatography

BACKGROUND: Among physicians who perform endoscopic retrograde cholangiopancreatography (ERCP), the relationship between procedure volume and outcome is unknown. OBJECTIVE: Quantify the ERCP volume-outcome relationship by measuring provider-specific failure rates, hospitalization rates, and other quality measures. RESEARCH DESIGN: Retrospective cohort. SUBJECTS: A total of 16,968 ERCPs performed by 130 physicians between 2001 and 2011, identified in the Indiana Network for Patient Care. MEASURES: Physicians were classified by their average annual Indiana Network for Patient Care volume and stratified into low (<25/y) and high (≥25/y). Outcomes included failed procedures, defined as repeat ERCP, percutaneous transhepatic cholangiography or surgical exploration of the bile duct≤7 days after the index procedure, hospitalization rates, and 30-day mortality. RESULTS: Among 15,514 index ERCPs, there were 1163 (7.5%) failures; the failure rate was higher among low (9.5%) compared with high volume (5.7%) providers (P<0.001). A second ERCP within 7 days (a subgroup of failure rate) occurred more frequently when the original ERCP was performed by a low-volume (4.1%) versus a high-volume physician (2.3%, P=0.013). Patients were more frequently hospitalized within 24 hours when the ERCP was performed by a low-volume (28.3%) versus high-volume physician (14.8%, P=0.002). Mortality within 30 days was similar (low=1.9%, high=1.9%). Among low-volume physicians and after adjusting, the odds of having a failed procedure decreased 3.3% (95% confidence interval, 1.6%-5.0%, P<0.001) with each additional ERCP performed per year. CONCLUSIONS: Lower provider volume is associated with higher failure rate for ERCP, and greater need for postprocedure hospitalization

Tissue-specific inducible expression of antimicrobial peptide genes in Drosophila surface epithelia

The production of antimicrobial peptides is an important aspect of host defense in multicellular organisms. In Drosophila, seven antimicrobial peptides with different spectra of activities are synthesized by the fat body during the immune response and secreted into the hemolymph. Using GFP reporter transgenes, we show here that all seven Drosophila antimicrobial peptides can be induced in surface epithelia in a tissue-specific manner. The imd gene plays a critical role in the activation of this local response to infection. In particular, drosomycin expression, which is regulated by the Toll pathway during the systemic response, is regulated by imd in the respiratory tract, thus demonstrating the existence of distinct regulatory mechanisms for local and systemic induction of antimicrobial peptide genes in Drosophila

Cross-species comparative analysis of Dicer proteins during Sindbis virus infection

In plants and invertebrates RNA silencing is a major defense mechanism against virus infections. The first event in RNA silencing is dicing of long double stranded RNAs into small interfering RNAs (siRNAs). The Dicer proteins involved in this process are phylogenetically conserved and have the same domain organization. Accordingly, the production of viral derived siRNAs has also been observed in the mouse, but only in restricted cell types. To gain insight on this restriction, we compare the dicing activity of human Dicer and fly Dicer-2 in the context of Sindbis virus (SINV) infection. Expression of human Dicer in flies inefficiently rescues the production of viral siRNAs but confers some protection against SINV. Conversely, expression of Dicer-2 in human cells allows the production of viral 21 nt small RNAs. However, this does not confer resistance to viral infection, but on the contrary results in stronger accumulation of viral RNA. We further show that Dicer-2 expression in human cells perturbs interferon (IFN) signaling pathways and antagonizes protein kinase R (PKR)-mediated antiviral immunity. Overall, our data suggest that a functional incompatibility between the Dicer and IFN pathways explains the predominance of the IFN response in mammalian somatic cells

Crystal Structure of Diedel, a Marker of the Immune Response of Drosophila melanogaster

Background: The Drosophila melanogaster gene CG11501 is up regulated after a septic injury and was proposed to act as a negative regulator of the JAK/STAT signaling pathway. Diedel, the CG11501 gene product, is a small protein of 115 residues with 10 cysteines. Methodology/Principal Findings: We have produced Diedel in Drosophila S2 cells as an extra cellular protein thanks to its own signal peptide and solved its crystal structure at 1.15 A ˚ resolution by SIRAS using an iodo derivative. Diedel is composed of two sub domains SD1 and SD2. SD1 is made of an antiparallel b-sheet covered by an a-helix and displays a ferredoxin-like fold. SD2 reveals a new protein fold made of loops connected by four disulfide bridges. Further structural analysis identified conserved hydrophobic residues on the surface of Diedel that may constitute a potential binding site. The existence of two conformations, cis and trans, for the proline 52 may be of interest as prolyl peptidyl isomerisation has been shown to play a role in several physiological mechanisms. The genome of D. melanogaster contains two other genes coding for proteins homologous to Diedel, namely CG43228 and CG34329. Strikingly, apart from Drosophila and the pea aphid Acyrthosiphon pisum, Diedel-related sequences were exclusively identified in a few insect DNA viruses of the Baculoviridae and Ascoviridae families. Conclusion/Significance: Diedel, a marker of the Drosophila antimicrobial/antiviral response, is a member of a small famil

Tension-Compression Loading with Chemical Stimulation Results in Additive Increases to Functional Properties of Anatomic Meniscal Constructs

Objective: This study aimed to improve the functional properties of anatomically-shaped meniscus constructs through simultaneous tension and compression mechanical stimulation in conjunction with chemical stimulation. Methods: Scaffoldless meniscal constructs were subjected to simultaneous tension and compressive stimulation and chemical stimulation. The temporal aspect of mechanical loadingwas studied by employing two separate five day stimulation periods. Chemical stimulation consisted of the application of a catabolic GAG-depleting enzyme, chondroitinase ABC (C-ABC), and an anabolic growth factor, TGF-b1. Mechanical and chemical stimulation combinations were studied through a full-factorial experimental design and assessed for histological, biochemical, and biomechanical properties following 4 wks of culture. Results: Mechanical loading applied from days 10–14 resulted in significant increases in compressive, tensile, and biochemical properties of meniscal constructs. When mechanical and chemical stimuliwere combined significant additive increases in collagen per wet weight (4-fold), compressive instantaneous (3-fold) and relaxation (2-fold) moduli, and tensile moduli in the circumferential (4-fold) and radial (6-fold) directions were obtained. Conclusions: This study demonstrates that a stimulation regimen of simultaneous tension and compression mechanical stimulation, C-ABC, and TGF-b1 is able to create anatomic meniscus constructs replicating the compressive mechanica

Lack of an Antibacterial Response Defect in Drosophila Toll-9 Mutant

Toll and Toll-like receptors represent families of receptors involved in mediating innate immunity response in insects and mammals. Although Drosophila proteome contains multiple Toll paralogs, Toll-1 is, so far, the only receptor to which an immune role has been attributed. In contrast, every single mammalian TLR is a key membrane receptor upstream of the vertebrate immune signaling cascades. The prevailing view is that TLR-mediated immunity is ancient. Structural analysis reveals that Drosophila Toll-9 is the most closely related to vertebrate TLRs and utilizes similar signaling components as Toll-1. This suggests that Toll-9 could be an ancestor of TLR-like receptors and could have immune function. Consistently, it has been reported that over-expression of Toll-9 in immune tissues is sufficient to induce the expression of some antimicrobial peptides in flies. These results have led to the idea that Toll-9 could be a constitutively active receptor that maintain significant levels of antimicrobial molecules and therefore provide constant basal protection against micro-organisms. To test theses hypotheses, we generated and analyzed phenotypes associated with a complete loss-of-function allele of Toll-9. Our results suggest that Toll-9 is neither required to maintain a basal anti-microbial response nor to mount an efficient immune response to bacterial infection

Long-Range Activation of Systemic Immunity through Peptidoglycan Diffusion in Drosophila

The systemic immune response of Drosophila is known to be induced both by septic injury and by oral infection with certain bacteria, and is characterized by the secretion of antimicrobial peptides (AMPs) into the haemolymph. To investigate other possible routes of bacterial infection, we deposited Erwinia carotovora (Ecc15) on various sites of the cuticle and monitored the immune response via expression of the AMP gene Diptericin. A strong response was observed to deposition on the genital plate of males (up to 20% of a septic injury response), but not females. We show that the principal response to genital infection is systemic, but that some AMPs, particularly Defensin, are induced locally in the genital tract. At late time points we detected bacteria in the haemolymph of immune deficient RelishE20 flies, indicating that the genital plate can be a route of entry for pathogens, and that the immune response protects flies against the progression of genital infection. The protective role of the immune response is further illustrated by our observation that RelishE20 flies exhibit significant lethality in response to genital Ecc15 infections. We next show that a systemic immune response can be induced by deposition of the bacterial elicitor peptidoglycan (PGN), or its terminal monomer tracheal cytotoxin (TCT), on the genital plate. This immune response is downregulated by PGRP-LB and Pirk, known regulators of the Imd pathway, and can be suppressed by the overexpression of PGRP-LB in the haemolymph compartment. Finally, we provide strong evidence that TCT can activate a systemic response by crossing epithelia, by showing that radiolabelled TCT deposited on the genital plate can subsequently be detected in the haemolymph. Genital infection is thus an intriguing new model for studying the systemic immune response to local epithelial infections and a potential route of entry for naturally occurring pathogens of Drosophila

Resveratrol Inhibits Inflammatory Responses via the Mammalian Target of Rapamycin Signaling Pathway in Cultured LPS-Stimulated Microglial Cells

Resveratrol have been known to possess many pharmacological properties including antioxidant, cardioprotective and anticancer effects. Although current studies indicate that resveratrol produces neuroprotection against neurological disorders, the precise mechanisms for its beneficial effects are still not fully understood. We investigate the effect of anti-inflammatory and mechamisms of resveratrol by using lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells.BV-2 cells were treated with resveratrol (25, 50, and 100 µM) and/or LPS (1 µg/ml). Nitric oxide (NO) and prostaglandin E2 (PGE2) were measured by Griess reagent and ELISA. The mRNA and protein levels of proinflammatory proteins and cytokines were analysed by RT-PCR and double immunofluorescence labeling, respectively. Phosphorylation levels of PTEN (phosphatase and tensin homolog deleted on chromosome 10), Akt, mammalian target of rapamycin (mTOR), mitogen-activated protein kinases (MAPKs) cascades, inhibitor κB-α (IκB-α) and cyclic AMP-responsive element-binding protein (CREB) were measured by western blot. Resveratrol significantly attenuated the LPS-induced expression of NO, PGE2, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nuclear factor-κB (NF-κB) in BV-2 cells. Resveratrol increased PTEN, Akt and mTOR phosphorylation in a dose-dependent manner or a time-dependent manner. Rapamycin (10 nM), a specific mTOR inhibitor, blocked the effects of resveratrol on LPS-induced microglial activation. In addition, mTOR inhibition partially abolished the inhibitory effect of resveratrol on the phosphorylation of IκB-α, CREB, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK).This study indicates that resveratrol inhibited LPS-induced proinflammatory enzymes and proinflammatory cytokines via down-regulation phosphorylation of NF-κB, CREB and MAPKs family in a mTOR-dependent manner. These findings reveal, in part, the molecular basis underlying the anti-inflammatory properties of resveratrol