Pathogen-associated molecular patterns (PAMPs) trigger host immune response by activating pattern recognition receptors like toll-like receptors (TLRs). However, the mechanism whereby several pathogens, including viruses, activate TLRs via a non-PAMP mechanism is unclear. Endogenous “inflammatory mediators” called damage-associated molecular patterns (DAMPs) have been implicated in regulating immune response and inflammation. However, the role of DAMPs in inflammation/immunity during virus infection has not been studied. We have identified a DAMP molecule, S100A9 , as an endogenous non-PAMP activator of TLR signaling during influenza A virus (IAV) infection. S100A9 was released from undamaged IAV-infected cells and extracellular S100A9 acted as a critical host-derived molecular pattern to regulate inflammatory response outcome and disease during infection by exaggerating pro-inflammatory response, cell-death and virus pathogenesis. Genetic studies showed that the DDX21-TRIF signaling pathway is required for S100A9 gene expression/production during infection. Furthermore, the inflammatory activity of extracellular S100A9 was mediated by activation of the TLR4-MyD88 pathway. Our studies have thus, underscored the role of a DAMP molecule (i.e. extracellular S100A9) in regulating virus-associated inflammation and uncovered a previously unknown function of the DDX21-TRIF-S100A9-TLR4-MyD88 signaling network in regulating inflammation during infection. The lung disease severity following influenza A virus (IAV) infection is dependent on the extent of inflammation in the respiratory tract. Severe inflammation in the lung manifests in development of pneumonia. Therefore, it is very critical to identify cellular factors and dissect the molecular/cellular mechanism controlling inflammation in the respiratory tract during IAV infection. Knowledge derived from these studies will be instrumental in development of therapeutics to combat the lung disease associated with IAV infection. Towards that end, in the current study we have identified a cellular factor S100A9 which is responsible for enhanced inflammation during IAV infection. In addition, we have characterized a signal transduction pathway involving various cellular receptors and signaling adaptors that are involved in mediating S100A9-dependent inflammatory response. Thus, our studies have illuminated a cellular/molecular mechanism that can be intervened by therapeutics to reduce and control IAV-associated lung inflammatory disease like pneumonia. Pathogen-associated molecular patterns (PAMPs) are molecular signatures of pathogens which facilitate induction of the host immune response Influenza A virus (IAV) is a negative-sense, single-stranded RNA virus that causes severe respiratory tract infection Innate immunity, comprised of antiviral activity  and a controlled inflammatory response, is critical host defense machinery for virus clearance and the resolution of virus-induced disease Activation of PRRs could serve as a double-edged sword: While operating as host defense factors, activated PRRs can also contribute to the progression of virus-induced disease. For example, although TLR4 is activated during IAV infection, studies with TLR4 KO mice have shown that TLR4 contributes to exacerbated lung disease and mortality in IAV-infected animals We are interested in understanding the role of secreted soluble factors  in viral innate immunity In the current study, we have identified extracellular S100A9 protein as a host-derived molecular pattern regulating the pro-inflammatory response, cell death, and pathogenesis during IAV infection. We also show that DDX21/TRIF and TLR4/MyD88 pathways are respectively required for S100A9 gene expression and activity. In addition, we have uncovered DDX21-TRIF-S100A9-TLR4-MyD88 signaling network as a critical regulator of inflammation. This network may also contribute to inflammation and disease during both infection-associated and noninfectious inflammatory diseases and disorders.Macrophages are essential immune cells that modulate host defense, inflammation, and disease pathogenesis during IAV infection. Macrophages are also the major cytokine- and chemokine-producing cells during IAV infection and thus contribute to lung tissue damage There have been no studies to date on the signaling mechanism that regulates gene expression of S100 family of proteins. We examined the signaling mechanism involved in S100A9 expression. We infected BMDMs derived from wild-type (WT), TLR2 knockout (KO), TLR4 KO and TRIF KO mice with IAV. At 24 h postinfection, we evaluated S100A9 levels in the medium. TLR2 and TLR4 were not involved, since comparable S100A9 secretion was noted in WT and TLR KO BMDMs . A similRecently, DEAD box proteins  having RNA helicase activity has been implicated in innate immunity Mouse alveolar macrophages (MH-S cell line) were transfected with DDX21-specific siRNA or control scrambled siRNA, after which these cells were infected with IAV. DDX21 expression was monitored by RT-PCR. We observed induction of DDX21 expression following IAV infection . The silIn the preceding studies, the high levels of S100A9 secretion during infection suggested that secreted extracellular S100A9 may have some role during IAV infection. Therefore, we focused on the role and function of extracellular S100A9 during IAV infection. Earlier studies have shown that the S100A9/S100A8 complex is required for optimal LPS-induced TLR4-dependent TNF-α (TNF) production in bone marrow cells (comprised of undifferentiated monocytes and DCs) Mouse (J774A.1) and human (U937 cells) macrophages were incubated with purified mouse or human S100A9 proteins, respectively for 6–12 h . After tin vitro and in vivoWe next examined the role of secreted S100A9 in eliciting a pro-inflammatory response during IAV infection. We used blocking antibody against S100A9 to neutralize the activity of extracellular (secreted) S100A9. Previously, it was shown that this blocking antibody specifically inhibited the activity of the secreted extracellular form of S100A9 both Our finding that S100A9 contributes to the pro-inflammatory response during IAV infection was validated by using BMDMs derived from S100A9 KO mice. WT and S100A9 KO BMDMs were infected with IAV, after which TNF and IL-6 levels in the medium supernatant were measured by ELISA. As compared to WT cells, there were significant reductions in IL-6  and TNF During these studies we observed that IAV replication (as deduced from IAV HA mRNA expression) was significantly reduced in S100A9 KO BMDMs compared to WT cells . This reIt is important to mention that we observed S100A9 production from IAV-infected BMDMs at 4 h postinfection  and thatMacrophages undergo apoptosis during IAV infection We treated J774A.1 and MH-S macrophages with purified S100A9 protein for 48 and 72 h, then examined the apoptotic status of cells by monitoring annexin V and PI staining. The apoptosis rate was calculated based on the number of annexin V positive/PI negative cells (denoting early apoptosis)+number of annexin V positive/PI-positive cells (denoting late apoptosis) per total number of cells. We noted apoptosis in S100A9 protein-treated mouse macrophages . The resSince IAV infection triggers S100A9 secretion, we next examined whether extracellular S100A9 has a role in apoptosis of IAV-infected macrophages. J774A.1 macrophages were infected with IAV for 48 h in the presence of either control antibody (control IgG) or S100A9 blocking antibody. Significantly diminished apoptosis (reduction of 27%) occurred in macrophages treated with S100A9 antibody . These rPrevious studies have found that optimal TLR4 activation by LPS in bone-marrow cells required the activity of extracellular S100A9/S100A8 complex Since MyD88 is one of the critical adaptors for activated TLR4, we next investigated the role of MyD88 by using MyD88 KO BMDMs. Incubation of WT and MyD88 KO BMDMs with purified S100A9 protein significantly reduced production of IL-6  and TNF We next studied the role of TLR4/MyD88 in stimulating the pro-inflammatory response following IAV infection. After WT, MyD88 KO, and TLR4 KO BMDMs were infected with IAV, IL-6 levels were assessed by ELISA. Our study revealed that TLR4/MyD88 is an essential regulator of pro-inflammatory response during IAV infection, since significant reduction in IL-6  and TNF Our study showed that extracellular S100A9 uses TLR4/MyD88 signaling for the pro-inflammatory response during IAV infection. TLR4 activation has been associated with apoptosis induction via various mechanisms, including activation of the pro-apoptotic function of NF-κB, modulation of tumor-suppresser expression or function etc We next examined the role of TLR4 and MyD88 in apoptosis induction during IAV infection. We infected WT and TLR4 KO BMDMs with IAV and evaluated apoptosis 48 h later. Apoptosis analysis by annexin V staining  and TUNEin vivo role of S100A9 in regulating innate response during IAV infection of the airway, we next evaluated S100A9 expression and its secretion in the IAV-infected mouse respiratory tract. Mice were intratracheally inoculated with IAV and, at 1–6 days postinfection, lungs were harvested. S100A9 mRNA expression in the lungs were analyzed by RT-PCR. S100A9 transcripts were observed in infected lungs but not in lungs from uninfected animals  and extracellular functions in vivo, since viral burden in the lung is directly proportional to the degree of pro-inflammatory (and apoptotic) response . However, neutralizing the activity of extracellular S100A9 with S100A9 blocking antibody did not alter IAV replication in vitro (in vivo (please see below). We therefore used anti-S100A9 blocking antibody to specifically inhibit the activity of extracellular S100A9 in mice. We have previously shown that anti-S100A9 blocking antibody has extracellular S100A9 blocking activity S. pneumoniae infection We used anti-S100A9 antibody instead of doing our in vitro  and in vInitially, we investigated the role of secreted S100A9 in regulating IAV susceptibility. For these studies, mice were i.p. injected with either control IgG or anti-S100A9 blocking antibody. One day later, mice were infected with IAV via intra-tracheal (I.T) inoculation. Survival of IAV-infected mice was monitored until 8 days postinfection. Blocking S100A9 activity significantly reduced the mortality of IAV-infected mice , demonstIn addition, inflammation was reduced following inhibition of extracellular S100A9 activity  and S10AWe have identified extracellular (secreted) S100A9 as a critical regulator of the pro-inflammatory response following IAV infection of macrophages. To examine the physiological role of secreted S100A9 in lung inflammation, we tested whether intratracheal (I.T.) administration of purified S100A9 protein would trigger a pro-inflammatory response in the lungs. Indeed, this led to production of TNF  and IL-6Based on this observation, we next examined the role of extracellular S100A9 in airway pro-inflammatory response following IAV infection. Mice were given i.p. injections of control IgG antibody or anti-S100A9 blocking antibody. At 1 d post-antibody treatment, mice were infected with IAV via I.T route. Levels of IL-6 and TNF in the lung were measured by ELISA. Extracellular S100A9 contributes to production of pro-inflammatory mediators during infection as evident from reduced TNF  and IL-6In order to provide evidence for direct neutralization of S100A9 activity in the airway following i.p. administration of S100A9 antibody, we administered S100A9 antibody (via i.p.) to mice and after one day (to exactly mimic IAV infection studies) mice were inoculated with S100A9 protein via I.T route. Significant inhibition in pro-inflammatory activity was noted in the presence of S100A9 antibody , which sex vivo experiment with BALF-associated cells derived from IAV infected mice administered (via i.p) with either control antibody or S100A9 blocking antibody. Significant reduction in IL-6 and TNF production from BALF cells was observed in S100A9 blocking antibody treated mice  infection model, we show release of S100A9 from “undamaged” cells during IAV infection; which triggered PRR  signaling. Surprisingly, we observed that extracellular (secreted) S100A9 regulates two key mechanisms that contribute to inflammation during IAV infection. These are pro-inflammatory cytokine production during early infection and induction of apoptosis. We also found that S100A9-mediated activation of the TLR4/MyD88 pathway resulted in increased inflammation, which culminated in exacerbated IAV pathogenesis. Thus, our study shows a role of “non-PAMP” PRR-activating DAMPs in modulating immunity and inflammation during virus infection. We also have identified DDX21-TRIF-S100A9-TLR4-MyD88 as a novel signaling “network” that regulates inflammation. It is possible that a similar pathway is used to promote disease during infection with other viruses including highly pathogenic RNA viruses like SARS, Ebola virus, Marburg virus.Based on our results, we propose a model  whereby Mortality among IAV-infected individuals is associated with pneumonia, a disease characterized by massive lung inflammation leading to tissue damage and endothelial barrier disruption, resulting in fluid leakage in the airway and the development of edema. Highly pathogenic IAV strains have greater propensity to launch a hyper-inflammatory response in the respiratory tract upon infection, culminating in the development of pneumonia. Among the cellular factors that regulate IAV-induced lung disease, TLR4 is a major contributor to susceptibility and exacerbated pathophysiology associated with IAV infection in vitro and in vivo. Although few studies have been done on the mechanism of S100 gene induction during biological responses, we have shown that the S100A9 gene is induced by the DDX21-TRIF pathway. These studies also demonstrated the critical function of the DDX family PRRs in regulating expression of a host factor (S100A9) that is not a cytokine (i.e. IFNs).Previous studies reported that S100A9-S100A8 complex optimizes LPS-mediated TLR4 activation We have determined that the DDX21-TRIF pathway is required for S100A9 gene expression. A recent study has shown that the DDX1-DDX21-DHX36/TRIF pathway triggers a type-I interferon response in myeloid dendritic cells (mDCs) during virus infection and treatment of cells with dsRNA (poly-IC) In accord with the previous studies We have also identified extracellular S100A9 as one of the host factors that regulate apoptosis during IAV infection. Apoptosis is a key contributor to pathogenesis and the pathology associated with IAV infection Two arms of viral innate immunity consist of antiviral and inflammatory responses. Our studies have indicated that extracellular and intracellular S100A9 may function differently in terms of innate immune response during IAV infection, whereby extracellular S100A9 modulates pro-inflammatory response  and intracellular S100A9 is involved in orchestrating antiviral response to reduce viral burden. The differential activity of extracellular vs. intracellular S100A9 protein has been noted previously Klebsiella pneumoniae infection, since enhanced bacterial dissemination, lung damage, and susceptibility was observed in mice deficient in S100A9 expression Klebsiella we speculate that IAV infection of S100A9 KO mice will result in diminished IAV replication and as a consequence, these mice will exhibit reduced inflammation, susceptibility and pathogenesis. We will conduct these studies in the future to elucidate the exact role (and underlying mechanism) of intracellular S100A9 in controlling IAV infection/replication.S100A9 confers protective immunity during Apart from macrophages, epithelial cells  also produced S100A9 upon IAV infection . InteresIn summary, we have identified extracellular S100A9 as a host-derived molecular pattern that regulates inflammation during virus infection. In addition, we have uncovered DDX21-TRIF-S100A9-TLR4-MyD88 as a novel signaling “network” that regulates inflammation. Future studies dealing with identification and characterization of host-derived molecular patterns (e.g. DAMPs) during virus invasion may lead to the development of measures to combat infection-associated inflammatory diseases.Animal studies were performed according to housing and care of laboratory animals guidelines established by National Institutes for Health. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of University of Texas Health Science Center at San Antonio. The Animal Welfare Assurance # is A3345-01.PR/8/34 (H1N1)] virus was grown in the allantoic cavities of 10-day-old embryonated eggs Influenza A  and lysed by sonication. Upon centrifuging the lysate at 55,000×g for 30 min at 4°C, the supernatant was collected. Recombinant His-Tag S100A9 was purified by using a nickel column. S100A9 bound to the column was incubated with 10 U of biotinylated thrombin (Novagen) (for 20 h at room temperature) to free S100A9 from its His-Tag. Recombinant S100A9 was then eluted with PBS. The digestion and elution processes were repeated one more time to cleave the remaining undigested recombinant proteins, and streptavidin-agarose (Novagen) was added to remove contaminating thrombin. Finally, the protein preparation was passed through a polymyxin B-agarose column  to remove endotoxins.Murine S100A9 neutralizing antibody purified IgG from the serum of S100A9 immunized rabbits was generated as described previously Recombinant proteins were prepared in Hank's buffered salt solution (HBSS) buffer. The absence of endotoxin contamination in antibody and protein preparations was confirmed using the limulus amebocyte assay (Cambrex).Taq polymerase, 10 pmol of each oligonucleotide primer, 1 mM MgCl2, and 100 µM deoxynucleotide triphosphates in a final reaction volume of 25 µl. Following amplification, the PCR products were analyzed on 1.5% agarose gel. Equal loading in each well was confirmed by analyzing expression of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used to detect the indicated genes by RT-PCR were:Total RNA was extracted using Tri Reagent (Invitrogen). cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR was done using 0.25 units of GAPDH forward, 5′-GTCAGTGGTGGACCTGACCT, GAPDH reverse, 5′-AGGGGTCTACATGGCAACTG,Mouse GAPDH forward, 5′-GCCAAGGTCATCCATGACAACTTTGG, Mouse GAPDH reverse, 5′-GCCTGCTTCACCACCTTCTTGATGTCMouse S100A9 forward, 5′-GTCCTGGTTTGTGTCCAGGT, Mouse S100A9 reverse, 5′-TCATCGACACCTTCCATCAAMouse DDX21 forward, 5′-GATCCCCCTAAATCCAGGAA, Mouse DDX21reverse, 5′-TTCGGAAGGCTCCTCTGTTAMouse TNF-α forward, 5′-CCTGTAGCCCACGTCGTAGC, Mouse TNF-α reverse, 5′-TTGACCTCAGCGCTGAGTTGMouse IL-6 forward, 5′-TTGCCTTCTTGGGACTGATGCT, Mouse IL-6 reverse, 5′-GTATCTCTCTGAAGGACTCTGGIAV HA forward, 5′- CCCAAGGAAAGTTCATGG, IAV HA reverse, 5′-GAACACCCCATAGTACAAGGU937 cells, alvelolar macrophages, BMDM, MH-S, and J774A.1 were infected with purified IAV [1 multiplicity of infection (MOI)−2 MOI as indicated] in serum-free, antibiotic-free OPTI-MEM medium (Gibco). Virus adsorption was done for 1.5 h at 37°C, after which cells were washed twice with PBS. Infection was continued in the presence of serum containing DMEM or RPMI medium for the specified time points.In some experiments, cells were infected in the presence of 2 ng–10 ng/ml control IgG  or 2 ng–10 ng/ml anti-S100A9 blocking antibody. Following virus adsorption, antibodies were added to the cells and the infection was carried out in the presence of the antibodies. In addition, in some experiments infection was done in the presence of purified S100A9 protein or HBSS buffer (vehicle control). Purified S100A9 protein (5 µg/ml) was added to S100A9 KO BMDMs following virus adsorption. Purified protein was present during infection.Control siRNA and mouse DDX21 siRNA were purchased from Santa Cruz Biotechnology. MH-S cells were transfected with 40 pmol of siRNAs using Lipofectamine 2000 (Invitrogen). At 48 h posttransfection, the cells were infected with IAV.Medium supernatant and mouse lung homogenate were analyzed for TNF and IL-6 levels by using a TNF and IL-6 specific ELISA kit . For S100A9 ELISA, Costar High-Binding 96-well plates  were coated overnight at 4°C with 800 ng/well of purified rabbit IgG against mouse S100A9 or 100 ng/well of goat polyclonal human S100A9 antibody (Abcam) diluted in 0.1 M carbonate buffer, pH 9.6. The wells were blocked with PBST+1% BSA for 1 h at room temperature. The samples were added and incubated overnight at 4°C. The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature. The plates were then washed three times in PBST. To detect mouse S100A9, rabbit anti-goat HRP (Bio-Rad) was added to the plates. To detect human S100A9, goat anti-mouse HRP (Bio-Rad) was added. After 1 h incubation at room temperature, the plates were washed three times with PBST. TMB-S substrate (100 µl/well) (Sigma-Aldrich) was added to the plates according to the manufacturer's instructions. The ODs were detected at 450 nm, using a Modulas micro-plate reader.To detect i.p.-injected S100A9 antibody in the lung homogenate, Costar High-Binding 96-well plates were coated overnight at 4°C with mouse S100A9 protein diluted in 0.1 M carbonate buffer, pH 9.6. The wells were blocked with PBST+1% BSA for 1 h at room temperature. The lung homogenate was added and incubated overnight at 4°C. The plates were washed three times with PBST and goat anti-rabbit HRP (Bio-Rad) was added. After 1 h of incubation at room temperature, the plates were washed three times with PBST. TMB-S substrate (100 µl/well) (Sigma-Aldrich) was added to the plates according to the manufacturer's instructions. ODs were detected at 450 nm by using a Modulas micro-plate reader.5 pfu/mouse) in 100 µl of PBS (Invitrogen). Control mice were sham-inoculated with 100 µl of PBS. Survival was monitored until 8 days postinfection. For pathogenesis assay, mice were inoculated with IAV (2×104 pfu/mouse via the I.T route) at 1 day after antibody treatment. At 3 days after infection, lungs and BALF were collected. Lung tissue sections were used for H&E analysis and in-situ TUNEL analysis. Lung homogenate was used for ELISA analysis (for TNF and IL-6). RT-PCR analysis for TNF and IL-6 expression was done with RNA isolated from mouse lungs. BALF was used for Western blotting with S100A9 antibody and S100A9 ELISA analysis.For survival experiments, 6–8-week old pathogen-free WT C57BL/6 mice (Jackson Laboratory) were injected i.p. with 2 mg/mouse of either control IgG or anti-S100A9 antibody. One day later, mice were anesthetized and inoculated via the intratracheal or I.T route with IAV  diluted in PBS or HBSS buffer diluted in PBS (vehicle control) was administered to mice via the I.T route. At 8 h posttreatment, TNF and IL-6 expression and production in the lung was monitored by RT-PCR and ELISA.Lung sections from mock- or IAV-infected mice were stained with goat anti-mouse S100A9 antibody (1∶100 dilution) (R&D) for 2 h at room temperature. After washing five times with PBS, lung sections were incubated with anti-goat Texas Red (1∶50 dilution) (Vector Labs) for 1 h at room temperature. After washing three times with PBS, sections were mounted with DAPI containing mounting solution (Invitrogen). Sections were visualized by fluorescence microscopy.http://rsbweb.nih.gov/ij/) as described previously by us 2 µm×4.09×102 µm of TUNEL-stained lung section was scanned by Image J software. Gross apoptotic area was expressed as pixels per micron. This value was used to calculate the percentage of the apoptotic area in each analysis. Three IAV- infected mice treated with control IgG and three IAV-infected mice treated with S100A9 antibody were used. Data were collected from 9 areas per mouse from each experimental group. The values obtained from the 27 lung section areas of each experimental group were used for statistical analysis.To study apoptosis in the respiratory tract, TUNEL assays were done. Formalin-fixed lungs from IAV-infected mice were used. The TUNEL assay was done using an ApopTag Peroxidase In Situ Apoptosis Detection Kit . Digital images of TUNEL-stained lung sections were examined by light microscopy. Digital images were used to count the number of TUNEL-positive cells, using Image J software from NIH (Hematoxylin and eosin (H&E) staining was performed on paraffin-embedded mouse lung sections. Briefly, slices of lung were sequentially rehydrated in 100% and 95% ethanol followed by xylene deparaffinization. After rinsing with distilled water, sections were stained with hematoxylin for 8 min and counterstained in eosin for 1 min followed by serial dehydration with 95% and 100% ethanol. Sections were then mounted on coverslips.IAV-infected and S100A9 protein-treated cells were examined for apoptosis by annexin V labeling, using an annexin V/propidium iodide (PI) apoptosis detection kit  Figure S1A) U937 and J774.1 cells were infected with IAV at 1 MOI and 2 MOI, respectively. At indicated post-infection time-periods the medium supernatant was collected to assess levels of LDH by Biovision LDH kit. The value shown represents the mean ± standard deviation from three independent experiments. (B) RT-PCR analysis of S100A9 expression in IAV infected MH-S cells transfected with either control siRNA or DDX21 siRNA. The RT-PCR data is a representative of three independent experiments with similar results. Method: LDH assay was performed by using the LDH-Cytotoxicity Assay kit-II . Briefly, medium supernatant from IAV-infected macrophages were collected and centrifuged at 600×g for 10 min. After centrifugation, clear medium supernatant was incubated with LDH Reaction Mix for 15 min at room temperature to assess levels of LDH by measuring the absorbance at 450 nm, using a Modulas micro-plate reader. Percentages of cytotoxicity was calculated according to manufacturer's instructions and values of background control (i.e. medium only) were subtracted from all other values.((PDF)Click here for additional data file.Figure S2A) RT-PCR analysis of IAV hemagglutinin (HA) expression in IAV infected MH-S cells transfected with either control siRNA or DDX21 siRNA. (B) RT-PCR analysis of IAV HA expression in infected wild type (WT) and TRIF knockout (KO) bone marrow derived macrophages (BMDM). The RT-PCR gel shown in (A) and (B) is a representative of three independent experiments with similar results. BMDMs isolated from WT and MyD88 KO (C) or MAVS KO (D) mice were infected with IAV. At 24 h post-infection time-period the medium supernatant was collected to assess levels of S100A9 protein by ELISA. The value shown in (C) and (D) represents the mean ± standard deviation from three independent experiments performed in triplicate. p value shown in the figure was derived by using Student's t test.((PDF)Click here for additional data file.Figure S3A) and mouse TNF-α(TNF) (B). Each RT-PCR data is a representative of three independent experiments with similar results. Vehicle; cells incubated with HBSS buffer (vehicle control).J774A.1 macrophages were incubated with purified recombinant mouse S100A9 protein (5 ug/mL) for 6 h and 12 h. RT-PCR analysis was performed to detect expression of mouse IL-6 ((PDF)Click here for additional data file.Figure S4A) and TNF (B) production by ELISA. (C) TNF production from mouse J774A.1 macrophages infected with IAV (2 MOI) in the presence of either control IgG (IgG) or anti-S100A9 blocking  antibody (S100A9 Ab) was analyzed by ELISA. (D) IL-6 production from wild-type BMDM infected with IAV in the presence of either control IgG or S100A9 Ab was analyzed by ELISA. (E) RT-PCR analysis of IL-6 expression in IAV infected BMDM treated with either control IgG or S100A9 Ab. The RT-PCR data is a representative of three independent experiments with similar results. The values shown in (A), (B), (C) and (D) represent the mean ± standard deviation from three independent experiments performed in triplicate. *p<0.05 using a Student's t test.Purified recombinant S100A9 protein was incubated with polymyxin B (10 µg/mL) for 2 h. S100A9 protein was also heat inactivated at 80°C for 30 min. Polymyxin treated and heat inactivated S100A9 protein (5 ug/mL) was then added to primary bone marrow derived macrophages (BMDM) to assess IL-6 ((PDF)Click here for additional data file.Figure S5A) RT-PCR analysis of IAV hemagglutinin (HA) expression in mouse J774A.1 macrophages infected with IAV (2 MOI) in the presence of either control IgG (IgG) or anti-S100A9 blocking  antibody (S100A9 Ab). (B) RT-PCR analysis of IAV HA expression in wild-type (WT) bone marrow derived macrophages (BMDM) infected with IAV in the presence of either control IgG or S100A9 Ab. (C) RT-PCR analysis of TNF-α expression in IAV infected WT and S100A9 knockout (KO) BMDMs. Each RT-PCR data is a representative of three independent experiments with similar results.((PDF)Click here for additional data file.Figure S6A) Primary bone marrow derived macrophages (BMDM) were treated with 5 µg/ml poly-IC . At indicated post-treatment time-periods the medium supernatant was collected to assess levels of S100A9 protein by ELISA. The values shown represent the mean ± standard deviation from three independent experiments performed in triplicate. *p<0.05 using a Student's t test. BMDM isolated from wild-type (WT) and S100A9 knockout (KO) mice were treated with poly-IC (10 µg/ml). At indicated post-treatment time-periods the medium supernatant was collected to assess levels of mouse IL-6 (B) and TNF-α (C) by ELISA. S100A9 KO BMDMs were treated with poly-IC (for 24 h) in the presence of purified recombinant mouse S100A9 protein (5 µg/ml). Medium supernatant was collected from treated cells to assess levels of mouse IL-6 (D) and TNF-α (E) by ELISA. Vehicle control cells (veh) were incubated with HBSS buffer. The values shown in (B), (C), (D) and (E) represent the mean ± standard deviation from three independent experiments performed in triplicate. *p<0.05 using a Student's t test. WT and S100A9 KO BMDMs were treated with either imiquimod (1 µg/ml for 12 h)  (F and G) or mouse TNF-α (10 ng/ml for 12 h)  (H). After treatment, the medium supernatant was collected to assess levels of mouse IL-6 (F and H) and TNF-α (G) by ELISA. The values shown in (F), (G) and (H) represent the mean ± standard deviation from three independent experiments performed in triplicate. No significant difference in WT vs. KO cells was observed. UT; untreated (i.e. treated with vehicle control). For poly-IC and imiquimod treatment, sterile endogenous-free water  served as the vehicle control; while PBS served as the vehicle control for TNF treatment.Role of S100A9 during pro-inflammatory response mediated by poly-IC, TNF and imiquimod. ((PDF)Click here for additional data file.Figure S7A) RT-PCR analysis of IAV HA expression in infected WT and S100A9 KO BMDM. RT-PCR data is a representative of three independent experiments with similar results. (B) Mouse J774A.1 macrophages were incubated with purified recombinant mouse S100A9 protein (5 µg/ml) for 48 h and 72 h. The apoptotic state of these cells was examined by TUNEL analysis. TUNEL positive cells were analyzed by image J software. Percent TUNEL positive cells denotes ratio of number of TUNEL positive cells/total number of cells. (C) Mouse alveolar macrophage MH-S cell-line was incubated with purified S100A9 protein (5 µg/ml). The apoptotic status was determined as described in (B). (D) Mouse J774A.1 macrophages were infected with IAV (2 MOI) in the presence of either control IgG (IgG) or anti-S100A9 blocking  antibody (S100A9 Ab). At 24 h post-infection, the apoptotic state of these cells was determined as described in (B). The values represents mean ± standard deviation from three independent experiments, *p<0.05 by Student's t test. Veh; cells incubated with HBSS buffer (vehicle control).((PDF)Click here for additional data file.Figure S8A) RT-PCR analysis of TNF expression in S100A9 protein treated wild-type (WT) and TLR4 knockout (KO) bone marrow derived macrophages (BMDM). (B) RT-PCR analysis of TNF expression in S100A9 protein treated WT and MyD88 KO BMDMs. (C) RT-PCR analysis of IL-6 expression in IAV infected WT and TLR4 KO BMDMs. (D) RT-PCR analysis of IAV hemagglutinin (HA) expression in IAV infected WT and TLR4 KO BMDMs. Each RT-PCR data is a representative of three independent experiments with similar results.((PDF)Click here for additional data file.Figure S9A) S100A9 production in the airway is not inhibited by S100A9 blocking antibody-Broncho-alveolar lavage fluid (BALF) isolated from IAV infected  mice administered with either control IgG (IgG) or anti-S100A9 blocking  antibody (S100A9 Ab)  were subjected to ELISA analysis to determine levels of S100A9 protein in BALF. The values represent the mean ± standard deviation from three independent experiments performed in triplicate. (B) Presence of S100A9 antibody in the lung of mice administered with S100A9 blocking antibody via intra-peritoneal route- Lung homogenate prepared from IAV infected  mice administered with either IgG or S100A9 Ab  were subjected to ELISA analysis to determine levels of S100A9 antibody. The values represent the mean ± standard deviation from three independent experiments performed in triplicate. (C) Body weight of IAV infected (100 pfu/mouse) mice (n = 7 mice/group) administered with either IgG or S100A9 Ab . The result represents mean ± s.e.m. p<0.05 (IgG treated mice vs. S100A9 Ab treated mice) based on Student's t test. The body weight of mock infected mice treated with either IgG or S100A9 Ab were monitored every 2 d (till 18 d post-antibody treatment). None of the mice lost weight (data not shown). Method: To detect inter-peritoneal administered S100A9 antibody in the lung homogenate, Costar High Binding 96-well plates  were coated overnight at 4°C with mouse S100A9 protein diluted in 0.1 M carbonate buffer, pH 9.6. The wells were blocked with PBST+1% BSA for 1 h at room temperature. The lung homogenate was added and incubated overnight at 4°C. The plates were then washed three times with PBST, followed by addition of goat anti-rabbit HRP (Bio-Rad). Following 1 h incubation at room temperature, the plates were washed three times with PBST. TMB-S substrate (100 µl/well) (Sigma-Aldrich) was added to the plates according to the manufacturer's instructions. The ODs were detected at 450 nm by using Modulas micro-plate reader.((PDF)Click here for additional data file.Figure S10A) A magnified version of the H&E stained lung section from B) Mice were administered with purified recombinant mouse S100A9 protein (15 µg/mouse) via intra-tracheal route. At 8 h post-administration, levels of mouse IL-6 in the lung was assessed by performing ELISA analysis with lung homogenate. The values represents mean ± standard deviation from three independent experiments, *p<0.05 by Student's t test. Veh; cells incubated with HBSS buffer (vehicle control). (C) H&E staining of lung sections from mice administered  with either vehicle or purified recombinant mouse S100A9 protein (15 µg/mouse).((PDF)Click here for additional data file.Figure S11A) Lung homogenate prepared from mock infected and IAV infected  mice administered with either control IgG (IgG) or anti-S100A9 blocking  antibody (S100A9 Ab)  were subjected to ELISA analysis to determine levels of mouse IL-6 in the lung. The values represents mean ± standard deviation from three independent experiments, *p<0.05 by Student's t test. (B) RT-PCR analysis of TNF expression in the lung of IAV infected mice treated with either control IgG or S100A9 Ab. RT-PCR data represents two mock mice/group and three infected mice/group and the data is a representative of three independent experiments with similar results.((PDF)Click here for additional data file.Figure S12A) Blocking extracellular S100A9 does not affect IAV burden in the lung - IAV infectious titer in the lung homogenate of infected mice  administered with either control IgG (IgG) or anti-S100A9 blocking  antibody (S100A9 Ab)  was assessed at 3 d post-infection by plaque assay analysis. The viral titer represents the mean ± standard deviation from three independent experiments performed in triplicate. p value (p = 0.1) shown in the figure was derived by using Student's t test. (B and C) Therapeutic activity of S100A9 blocking antibody – (B) Lung homogenate prepared from mock infected and IAV infected  mice administered with either IgG or S100A9 Ab  were subjected to ELISA analysis to determine levels of mouse TNF-α in the lung. The values represents mean ± standard deviation from three independent experiments, *p<0.05 by Student's t test. (C) H&E staining of lung sections from mock infected and IAV infected mice administered with either IgG or S100A9 Ab .((PDF)Click here for additional data file.Figure S13A) S100A9 antibody administered via intra-peritoneal route blocks pro-inflammatory activity of S100A9 in the airway - Mice were administered with either control IgG (IgG) or anti-S100A9 blocking  antibody (S100A9 Ab) (2 mg of antibody/mouse was administered via i.p route). After 24 h, mice were administered with purified recombinant mouse S100A9 protein (15 µg/mouse) via intra-tracheal route. At 8 h post-S100A9 protein administration, levels of mouse TNF-α in the lung was assessed by performing ELISA analysis with lung homogenate. The values represents mean ± standard deviation from three independent experiments, *p<0.05 by Student's t test. (B) S100A9 production from MH-S cells infected with either UV-IAV (UV-irradiated IAV virus) or control-IAV (non UV irradiated IAV virus) for 12 h and 24 h. S100A9 was measured by ELISA analysis of medium supernatant. The values represent the mean ± standard deviation from two independent experiments performed in triplicate,*p<0.05 using a Student's t test.((PDF)Click here for additional data file.Figure S14Method: Lung  epithelial cells were isolated from mouse lung as described previously . Sternohyoid muscles of the neck were removed from anesthetized mice to expose the trachea. The lungs were perfused (by using barrel-tip needle) with 6 ml of sterile PBS. The enzyme dispase (3 ml) (StemCell Technologies) was instilled through a cannula in the trachea, followed by injection of 1 ml of 1% low melt agarose. Following 2 min incubation of the mice on ice (to solidify the agarose), the lungs were removed and further incubated with 1 ml dispase for 45 min (at room temperature). The lungs were then transferred to a culture dish containing 8 ml DMEM medium (with 20 mM HEPES and 100 units/ml of penicillin and streptomycin). The lungs were then gently teased away from cell suspension and agitated for 10 min on ice. The cell suspension was filtered successively through 100 µm, 40 µm, and 20 µm nylon gauze. The suspension was then centrifuged for 5 min at 130 g. The cell pellet was re-suspended in complete DMEM medium , penicillin, streptomycin, and glutamine). The re-suspended cells were added to dishes pre-coated with 45 µg/ml of anti-mouse CD45  and CD16/32  antibodies. After 2 h incubation (at 37°C in the CO2 incubator), the non-adherent cells were collected and centrifuged for 5 min at 130 g. The cell pellet was re-suspended in complete DMEM and transferred to a six-well plate. Light microscopy showed that the adhered cells possess epithelial morphology. These cells which constitute primary lung  epithelial cells were infected with IAV (2 MOI) and medium supernatant was collected to assess S100A9 levels by ELISA.Release of S100A9 protein from IAV infected primary mouse lung epithelial cells. Mouse epithelial cells isolated from the lung of C57BL/6 mice were plated and infected with IAV. At 8 h and 16 post-infection, medium supernatant was collected to assess levels of S100A9 protein by ELISA. The values shown represent the mean ± standard deviation from three independent experiments performed in triplicate. *p<0.05 using a Student's t test. (PDF)Click here for additional data file.Table S1Extracellular vs. intracellular S100A9 protein levels in influenza A virus (IAV) infected bone marrow derived macrophages. S100A9 protein levels were measured by ELISA. The values represent the mean ± standard deviation from three independent experiments performed in triplicate. The levels of extracellular (medium supernatant) vs. intracellular (cell lysate) S100A9 was compared. Significantly less  S100A9 was present in the medium supernatant compared to the cell lysate. Percent extracellular vs. intracellular was calculated based on the ratio of S100A9 protein levels in medium supernatant or cell lysate/total S100A9 protein (i.e. the sum of S100A9 present in medium supernatant+S100A9 present in the cell lysate). We failed to detect any extracellular (in the medium supernatant) S100A9 in mock infected macrophages (data not shown).(PDF)Click here for additional data file.


MEN1 or RET, respectively. Recently, mutations in CDKN1B, encoding the cyclin-dependent kinase (Cdk) inhibitor p27, were identified in patients having a MEN1-like phenotype but no MEN1 gene mutations. Currently, the molecular mechanisms mediating the role of p27 in tumor predisposition are ill defined. We here report a novel germline missense variant in CDKN1B  found in a patient with multiple endocrine tumors. We previously reported a nonsense p27 mutation  in two patients with MEN1-like phenotype. Functional assays were used to characterize p27P69L and p27W76X in vitro. We show that p27P69L is expressed at reduced level and is impaired in both binding to Cdk2 and inhibiting cell growth. p27W76X, which is mislocalized to the cytoplasm, can no longer efficiently bind Cyclins-Cdks, nor inhibit cell growth or induce apoptosis. In the patient's tumor tissues, p27P69L associates with reduced/absent p27 expression and in one tumor with loss-of-heterozygosity. Our results extend previous findings of CDKN1B mutations in patients with MEN1-related states and support the hypothesis of a tumor suppressor role for p27 in neuroendocrine cells. © 2010 Wiley-Liss, Inc.Multiple endocrine neoplasia (MEN) syndromes are characterized by tumors involving two or more endocrine glands. Two MEN syndromes have long been known: MEN1 and MEN2, caused by germline mutations in  Cdkn1b (MIM# 600778), which encodes the p27 cyclin-dependent kinase (Cdk) inhibitor, causes a multiple endocrine neoplasia (MEN)-like syndrome in the rat  in a female patient with small-cell neuroendocrine cervical carcinoma, Cushing's disease, and hyperparathyroidism  were identified in patients with hereditary predisposition to MEN1-like tumors or primary hyperparathyroidism (1°HPT) , while p27P95S showed decreased binding affinity for Grb2, an adaptor protein involved in the activation of Ras signalling . Due to the small number of mutation-bearing patients so far reported, the phenotypic features associated to MEN4 are still undefined. Moreover, the limited molecular characterization of the naturally-occurring CDKN1B mutations identified hinders a thorough understanding of the role of p27 in neuroendocrine tumor predisposition.We recently demonstrated that germline mutation in  the rat . Followin humans . Subsequyroidism . This mu (1°HPT) . In vitrgnalling . TherefoCDKN1B mutations and, if found, to functionally characterize them in vitro. We report here a novel CDKN1B mutation found in a patient with multiple endocrine tumors. Functional analysis of two mutated p27 proteins (the novel we identified and one we previously reported) regarding subcellular localization, protein binding and degradation kinetic, growth suppression and apoptosis are presented.The aim of our study was to search for new MEN1 gene mutation, were screened for mutations in CDKN1B (GenBank entry NM_004064.3) as previously reported (http://www.hgvs.org/mutnomen). The initiation codon is codon 1.The study was approved by the Ethics Committee of the University of Ferrara, Italy and informed written consent was obtained from all patients. Twenty-seven Italian patients displaying a MEN1-like phenotype , but lacking a reported . To detereported . Loss ofreported  using thCDKN1B cDNA cloned in a pEYFP backbone, kindly provided by J. Slingerland . Primary antibodies used were: anti-p27 (clone 57) and anti-GFP tag (BD Biosciences); anti-Skp2 (H-345); anti-Cdk2 (D-12); anti-Cdk4 (C-22); anti-CyclinD1 (H-295); anti-Cyclin E (M-20); anti-α-tubulin .The two described mutations were introduced by site-directed mutagenesis  in the wild-type human HeLa and MCF7 cell lines were respectively maintained in DMEM or RPMI 1640 media supplemented with 10% fetal bovine serum, 20 mM L-glutamine, 100 units/ml of penicillin G sodium, and 100 µg/ml streptomycin . The GH3 cell line was maintained in F-12K medium supplemented with 15% horse serum, 2.5% fetal bovine serum, 20 mM L-glutamine, 100 units/ml of penicillin G sodium, and 100 µg/ml streptomycin.For p27 half-life determination in exponentially growing cells, cycloheximide  was added to the cells for the indicated times.Transfections were carried out as already reported . When esSKP2  and KPC1  and scrambled siRNA (Ambion) were obtained and transfected into MCF7 cells using X-tremeGENE reagent (Roche) together with 0.03 µg of plasmid DNA.For RNAi studies, short interfering RNA (siRNA) duplexes specific for Protein extraction and immunoblotting were carried out as already reported .For IP, 500 µg of total protein were incubated with 5 µl of anti-YFP tag antibody (Clontech-Takara Bio Europe) and 20 µl of agarose resin (Santa Cruz) overnight at 4°C. For GST-pull down assay, 500 µg of total protein were incubated overnight at 4°C with 2 µg of recombinant Cdk2-GST (Abnova) and 20 µl of Glutathione Agarose (Santa Cruz).p27 knock-out (-/-) MEFs were obtained from 13 days old embryos upon trypsin digestion for 15 min at 37°C. Cells were grown in DMEM supplemented with 10% fetal bovine serum, 20 mM L-glutamine, 100 units/ml of penicillin G sodium, and 100 µg/ml streptomycin . For localization experiments, MEFs were grown on coverslips, transfected and processed for immunocytochemistry as described  with minTo examine clonogenic activity, GH3 cells were plated  in 6-well plates and transfected with p27wt, p27W76X or p27P69L using Fugene-6 (Roche). The day after the transfection, the cells were diluted 1:6 and, starting from the day after, selected for 6 weeks by adding 0.4 mg/ml G418 to the culture medium and stained with 0.3% crystal violet in 30% ethanol. Colonies that contained ≥50 cells were scored.p27W76X GH3 stable clones, and untrasfected GH3 cells were plated in duplicate  in 12-well plate, in full medium plus 400 µg/ml G418. The number of cell was counted every two days over a 9 day-period using a cell counter.CDKN1B cDNA cloned into the pEYFP  backbone. Immunohistochemistry were performed using a monoclonal anti-p27 antibody (BD Biosciences) as already reported  in patient FL, a 79-year-old Caucasian female  in a proband with GH-secreting pituitary adenoma and 1°HPT, and in her sister displaying renal angiomyolipoma . Upon trin vitro and showed that p27P69L is consistently expressed at reduced level compared to p27wt in all cell lines , p27P69L localized mainly to the nucleus, like p27wt , althougp27P69L is consistently expressed at lower steady-state level compared to p27wt. This could be due to enhanced protein degradation, the mechanism responsible for p27 down-regulation in many human tumors . ExponenKPC1, play a role also in the degradation of p27P69L and p27W76X, respectively    showed tockdown) . Co-tranolecules .Considering the crystal structure of the p27-CyclinA-Cdk2 protein complex, the P69L change affects one of the six amino acids involved in the direct binding to Cdk2 . To verip27W76X retains most of the cyclin/Cdk binding domain of p27 (aminoacids 28 to 90), therefore it could bind to and sequester in the cytoplasm the canonical p27 partners: Cyclins E and D1, Cdk2 and Cdk4. Upon transfection of proliferating HeLa cells, we immunoprecipitated (IP) p27W76X and detected interacting Cyclins and Cdks by immunoblotting with specific antibodies. A minimal fraction of p27W76X binds to Cyclin D1, while there is no association with the other molecules . Similarp27 is a negative regulator of cell cycle progression, and its loss is associated with disease progression and unfavourable outcome in many cancer types . To deteTo verify that the long-term expression of p27W76X has no effect on cell proliferation, we picked and expanded several stably-transfected, GH3-derived clones. Selected representative clones were employed for growth curves over a period of 9 days. In agreement with the clonogenic assay results, constitutive overexpression of p27W76X did not affect GH3 cell proliferation .Tumor suppression by p27wt works through inhibition of cell proliferation, but also induction of apoptosis in some cell types . We asseWe analyzed the expression of p27 in the parathyroid adenoma and one bronchial carcinoid of the patient carrying the p27P69L mutation using IHC. Parathyroid adenoma cells showed weak p27 nuclear staining in <1% of the tumor cells  . The parCDKN1B germline mutations in a sub-set of patients with multiple endocrine tumors. We identified a novel germline missense mutation in a patient with multiple endocrine and non-endocrine malignancies. Based on few recent studies the incidence of CDKN1B mutations in patients with a MEN 1-related phenotype is estimated to be 1.5%-2.8% (CDKN1B mutations to the MEN4 syndrome.Our current study confirms and extends previous findings concerning the presence of .5%-2.8% . In our in vitro characterization of the newly identified mutation (P69L), as well as of the W76X change, identified an altered molecular phenotype for both encoded proteins.Functional http://www.expasy.org/spdbv/), thereby dramatically altering the environmental conditions necessary for binding to the kinase. Our studies demonstrated that indeed this variant does not bind to Cdk2. The CyclinE/Cdk2 complex is a very important target of p27 and this binding is crucial for p27-mediated regulation of G1 to S phase cell cycle progression (in vitro.The P69L mutation affects an amino acid located in the Cdk2 binding domain of the protein (aminoacids 52-93) and this change is predicted with a high degree of confidence to reduce by more than 55% the hydrophobicity of the region  tumor suppressor . This goes beyond the low expression level of p27P69L mutation . The obsppressor , as formppressor . In contCDKN1B mutations show variable age at onset. The two probands carrying the W76X or the c.59_77dup, p.K25fs mutation developed tumors at relatively young age , while all other carriers showed a clinical phenotype at age 50 or older. We speculate that the age at onset of the mutation-carriers might be related to how severely the CDKN1B mutation alters the p27 sequence and likely its function, which would in turn provide stronger selective advantage and lead to cell expansion.The patient carrying the P69L mutation shows a relatively late age at onset of endocrine disease. The few patients so far reported who carry germline in vitro upon transfection but also, and more significantly, in the patient's normal kidney tissue by immunohistochemistry. Although mutated alleles encoding for truncated peptides tend to be unstable and often the encoded peptides are not detected, it has been demonstrated that p27 deletion mutants comprising only the N-terminus of the protein can be expressed in vitro (The W76X mutation prevents the encoded p27 protein from entering the nucleus, the cell compartment where p27 exerts its function as a cell cycle inhibitor, and indeed p27W76X can no longer inhibit the growth of GH3 cells both by clonogenic assay and by stable expression. Moreover, p27W76X has lost the pro-apototic function of p27wt. This is in agreement with reports showing that cells with cytoplasmic p27 are resistant to apoptosis, but can become sensitive upon relocalization of the protein to the nucleus . Interesin vitro .Although the W76X mutation alters several p27 functions, the only tumor tissue of a mutation-positive patient available for analysis showed no p27 immunoreactivity  while thCDKN1B mutations will help us understand the link between p27 and neuroendocrine tumor predisposition, a prerequisite to offer in the future personalized care to the families carrying such mutations.Studies on naturally-occurring


Both IgA and IgG antibodies are known to play important roles in protection against influenza virus infection. While IgG is the major isotype induced systemically, IgA is predominant in mucosal tissues, including the upper respiratory tract. Although IgA antibodies are believed to have unique advantages in mucosal immunity, information on direct comparisons of the in vitro antiviral activities of IgA and IgG antibodies recognizing the same epitope is limited. In this study, we demonstrate differences in antiviral activities between these isotypes using monoclonal IgA and IgG antibodies obtained from hybridomas of the same origin. Polymeric IgA-producing hybridoma cells were successfully subcloned from those originally producing monoclonal antibody S139/1, a hemaggulutinin (HA)-specific IgG that was generated against an influenza A virus strain of the H3 subtype but had cross-neutralizing activities against the H1, H2, H13, and H16 subtypes. These monoclonal S139/1 IgA and IgG antibodies were assumed to recognize the same epitope and thus used to compare their antiviral activities. We found that both S139/1 IgA and IgG antibodies strongly bound to the homologous H3 virus in an enzyme-linked immunosorbent assay, and there were no significant differences in their hemagglutination-inhibiting and neutralizing activities against the H3 virus. In contrast, S139/1 IgA showed remarkably higher cross-binding to and antiviral activities against H1, H2, and H13 viruses than S139/1 IgG. It was also noted that S139/1 IgA, but not IgG, drastically suppressed the extracellular release of the viruses from infected cells. Electron microscopy revealed that S139/1 IgA deposited newly produced viral particles on the cell surface, most likely by tethering the particles. These results suggest that anti-HA IgA has greater potential to prevent influenza A virus infection than IgG antibodies, likely due to increased avidity conferred by its multivalency, and that this advantage may be particularly important for heterosubtypic immunity. It is known that both IgA and IgG antibodies play important roles in protection against influenza virus infection Influenza A viruses are divided into subtypes based on the antigenicity of two envelope glycoproteins, hemagglutinin (HA) and neuraminidase (NA). To date, H1-H16 HA and N1–N9 NA subtypes have been found in wild aquatic birds, the natural reservoir of influenza A viruses We previously reported that an HA-specific monoclonal antibody (MAb) S139/1 (IgG) that originated from mice immunized with a virus of subtype H3 showed broad cross-reactivity to viruses with multiple HA subtypes, including H1, H2, H3, H13, and H16 Influenza A virus strains, A/Aichi/2/1968 (H3N2) (Aichi/H3), A/WSN/1933 (H1N1) (WSN/H1), A/Adachi/2/1957 (H2N2) (Adachi/H2), and A/gull/Maryland/704/1977 (H13N6) (Maryland/H13), were kindly provided by Dr. H. Kida, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. These viruses were propagated in the allantoic cavity of 10 day-old embryonated chicken eggs at 35°C for 48 hours. Virus particles were concentrated and purified by high-speed centrifugation of the allantoic fluid passed through a 10–50% sucrose density gradient. Purified viruses were disrupted with 50 mM Tris-HCl (pH 7.8) containing 0.5% Triton X-100 and 0.6 M KCl, and used as antigens for enzyme-linked immunosorbent assay (ELISA) 2. At 70% to 90% confluence, culture supernatants were screened for IgA production by sandwich ELISA using immunoplates previously coated with goat anti-mouse IgA (BETHYL). Cells from positive wells were harvested and replated at 10–100 cells per well. Screening for IgA and replating were repeated several times and IgA-producing cells were finally established from a single cell clone. Messenger RNA was extracted from the variant hybridoma clone using an RNeasy Mini Kit (QIAGEN). The variable region gene was amplified by reverse transcription and polymerase chain reaction using a One-Step PCR Kit (QIAGEN) with primers 5′-GAT GGT GGG ATT TCT CGC AGA CTC-3′ and 5′- SAR GTN MAG CTG SAG SAG TC-3′ for the heavy chain, and 5′- GGA TAC AGT TGG TGC AGC ATC-3′ and 5′- GAY ATT GTG MTS ACM CAR WCT MCA-3′ for the light chain, followed by direct sequencing. We confirmed that the amino acid sequence of the variable region of S139/1-derived IgA was identical to that of the original S139/1 IgG To obtain hybridoma variants producing p-IgA antibodies recognizing the same epitope as S139/1 IgG, we modified a sib selection/ELISA method that relied on spontaneous class-switching of the cells Hybridoma cells producing S139/1 IgA and IgG antibodies were cultured in BD Cell MAb Medium, Serum Free using CELLine Flask (BD). S139/1 IgA and IgG antibodies were purified from culture supernatants using KAPTIV-AE™ (TECNOGEN) and Protein G Sepharose 4 Fast Flow , respectively, according to the manufacturers' instructions. MAb concentrations were measured by optical density at 280 nm using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). Purified MAbs were analyzed by SDS-PAGE under reducing or nonreducing conditions. For reducing conditions, MAbs were mixed with SDS-PAGE sample buffer with 2-mercaptoethanol (2-ME) (Wako) and heated at 98°C for 5 minutes. For nonreducing conditions, MAbs were mixed with SDS-PAGE sample buffer without 2-ME and incubated at room temperature for 30 minutes. Protein bands stained with Quick-CBB PLUS (Wako) were quantified using a VersaDoc™ Imaging System (Bio-Rad) and Image Lab™ software (Bio-Rad).50) to influenza A virus antigens were determined using SoftMax® Pro 6.2.1 software (Molecular Devices) Binding of MAbs was measured by ELISA as described previously HI activities of the purified MAbs were tested by the standard method using 0.5% chicken erythrocytes. Neutralizing activities of MAbs were evaluated by the standard procedure of plaque-reduction tests using MDCK cells. Fourfold serial dilutions of MAbs (50 µl) were mixed with an equal volume of diluted virus solution (approximately 50–100 plaque-forming units), and incubated for 1 hour at room temperature. Then the mixture was inoculated onto a monolayer of MDCK cells on 12-well tissue culture plates. After 1-hour incubation at 35°C, the inoculum was aspirated and cells were washed once with serum-free MEM and overlaid with MEM containing 1% Bacto-agar and trypsin (5 µg/ml) (GIBCO). Plaques were counted after incubation at 35°C for 1 day (WSN/H1 and Adachi/H2), 2 days (Aichi/H3), or 3 days (Maryland/H13).MDCK cells on 24-well plates were first inoculated with viruses at a multiplicity of infection of 1-2, followed by incubation for 1 hour at 35°C. The inoculum was aspirated, and the cells were washed three times and cultured with MEM containing S139/1 IgG or IgA antibodies (0.1 or 1 µg/ml) for 12 hours. Culture supernatants were collected at 0, 6, and 12 hours after infection, and mixed with SDS-PAGE sample buffer with 2-ME and treated at 98°C for 5 minutes. After 10% SDS-PAGE, separated proteins were blotted on a polyvinylidene difluoride membrane (Millipore). Chicken antiserum to Aichi/H3 was used as the primary antibody to detect viral proteins. The bound antibody was detected with peroxidase-conjugated rabbit anti-chicken IgY (IgG) (H+L) , followed by visualization with Immobilon Western (Millipore). Band intensities of the M1 protein were analyzed with a VersaDoc™ Imaging System (Bio-Rad) and Image Lab™ software (Bio-Rad).Transmission electron microscopy (TEM) was carried out as described previously We first analyzed the purity and the molecular weight of purified S139/1 IgA and IgG antibodies by SDS-PAGE . Two pro50 values  50 values . In contrast, there were remarkable differences between S139/1 IgA and IgG antibodies in reactivities to the WSN/H1, Adachi/H2, and Maryland/H13 antigens. IgA bound to these heterologous antigens at an extent similar to that with the homologous H3 antigen, and the EC50 values for WSN/H1, Adachi/H2, and Maryland/H13 were only 2- to 5-fold lower than that for the Aichi/H3 antigen. However, IgG reactivities to the heterologous HAs were uniformly much lower than that to the H3 antigen as indicated by the considerably larger EC50 values for WSN/H1, Adachi/H2, and Maryland/H13  than for Aichi/H3 (0.0017 µg/ml).We then investigated the binding activities of S139/1 IgA and IgG antibodies to the homologous HA antigen  and heterologous HA antigens  by ELISA , and det0 values . EC50 isWe next compared HI activities of S139/1 IgA and IgG antibodies . Consist50) of S139/1 IgA and IgG were determined , although both S139/1 IgA and IgG antibodies exhibited less neutralizing activity for heterologous viruses than for Aichi/H3.We further compared the neutralizing activities of S139/1 IgA and IgG antibodies by the standard plaque reduction test  and the termined . Consisttermined , both S1To compare the abilities of S139/1 IgA and IgG antibodies to suppress the extracellular release of virus particles from infected cells To gain insight into the mechanism of IgA-mediated inhibitory effects on the viral release from infected cells, we used electron microscopy to examine Aichi/H3-infected MDCK cells cultured with S139/1 IgA or IgG. After 8-hour incubation, tight aggregation and abnormal accumulation of unreleased virus particles were found on the virus-infected cells cultured in the presence of S139/1 IgA , which wIt has been reported that intranasal immunization induces a more efficient cross-protective immune response against influenza virus than systemic immunization, and IgA antibodies are suggested to play a major role in antibody-mediated heterosubtypic immunity In ELISA, while S139/1 IgG bound strongly to the homologous Aichi/H3 HA, its cross-binding activity to heterologous HAs  was appreciably lower, in accord with previous studies As evidenced by the well-known protective efficacy of influenza virus NA inhibitors, the viral budding/release process is a promising target for antiviral development Based on this hypothesis, the epitope may also be an important factor, since efficient crosslinking is likely done by antibodies that target the globular head region of HA. Accordingly, S139/1 recognizes highly conserved residues in the receptor binding site of the HA molecule, and thus has strong neutralizing activity The present study demonstrates that anti-HA S139/1 IgA has greater antiviral potential against influenza A virus infection in vitro than IgG, and the advantage of IgA is more prominent in heterosubtypic cross-reactivity. The polymeric structure might be important for the enhanced ability of IgA antibodies. In addition, our data suggest that tethering and depositing newly budded virus particles on the infected cell surface may generally be one of the antibody-mediated protective mechanisms against enveloped viruses and emphasize the importance of p-IgA in mucosal immunity.Figure S1Competitive antibody binding assay using S139/1 IgA and IgG. ELISA plates were coated with the disrupted virus antigens (Aichi/H3), followed by blocking with 3% skim milk in PBS. Tenfold serially diluted S139/1 IgG and IgA were plated as competitive antibodies, followed by incubation with S139/1 IgA and IgG (1 ng/ml), respectively. Bound IgA and IgG were detected using goat anti-mouse IgA (α) and goat anti-mouse IgG (γ) antibodies conjugated to horseradish peroxidase. The reaction was visualized by adding 3,3′,5,5′-tetramethylbenzidine and the absorbance at 450 nm was measured.(TIFF)Click here for additional data file.Figure S2TEM images of Aichi/H3-infected MDCK cells cultured in the presence of MAb S139/1 IgA. MDCK cells infected with Aichi/H3 at a multiplicity of infection of 1-2 were incubated for 12 hours in the presence of S139/1 IgA. Ultrathin sections were examined by TEM. TEM images are shown at low (A and B) and high (C and D) magnifications.(TIFF)Click here for additional data file.


TOR1A , the gene responsible for the most common primary hereditary dystonia, encodes torsinA, an AAA ATPase family protein. However, the function of torsinA has yet to be fully understood. Here, we have generated and characterized a complete loss-of-function mutant for dtorsin, the only Drosophila ortholog of TOR1A. Null mutation of the X-linked dtorsin was semi-lethal with most male flies dying by the pre-pupal stage and the few surviving adults being sterile and slow moving, with reduced cuticle pigmentation and thin, short bristles. Third instar male larvae exhibited locomotion defects that were rescued by feeding dopamine. Moreover, biochemical analysis revealed that the brains of third instar larvae and adults heterozygous for the loss-of-function dtorsin mutation had significantly reduced dopamine levels. The dtorsin mutant showed a very strong genetic interaction with Pu (Punch: GTP cyclohydrolase), the ortholog of the human gene underlying DYT14 dystonia. Biochemical analyses revealed a severe reduction of GTP cyclohydrolase protein and activity, suggesting that dtorsin plays a novel role in dopamine metabolism as a positive-regulator of GTP cyclohydrolase protein. This dtorsin mutant line will be valuable for understanding this relationship and potentially other novel torsin functions that could play a role in human dystonia.Dystonia represents the third most common movement disorder in humans. At least 15 genetic loci (DYT1-15) have been identified and some of these genes have been cloned.  TOR1A (formerly DYT1) and displays dominant inheritance with reduced penetrance TOR1A encodes torsinA, a 332 amino acid protein from the AAA ATPase family. TorsinA is localized in the lumen of the endoplasmic reticulum and the nuclear envelope Dystonia is a movement disorder characterized by sustained muscle contraction. While 15 different gene loci have been implicated in primary hereditary dystonia (DYT1-DYT15) TOR1A, TOR1B, TOR2A, and TOR3A) have been described, while Drosophila melanogaster has only a single torsin gene, dtorsin (previously known as torp4a: CG3024) in 4C11 on the X chromosome dtorsin-encoded protein, Dtorsin, comprises 339 amino acids with 31.9% identity to human torsinA and also displays the characteristic features of the AAA ATPase gene family Drosophila of the mutant form of human torsinA, pan-neuronally or in monoaminergic cells results in a locomotor defect dtorsin gene in the Drosophila eye caused progressive degeneration of the retina, but no loss-of-function mutants of dtorsin have previously been characterized.In mammals, four paralogous torsin genes  the vast majority of mutant males died by the pre-pupal stage; 2) the few surviving adult males were sterile with reduced pigmentation and thin, short bristles; 3) the late third instar male larvae exhibited locomotor deficits. As some of these phenotypes are associated with mutations that disrupt dopamine metabolism dtorsin, we generated a loss-of-function mutation using ‘ends-out’ gene targeting designed to remove almost the entire coding sequence white+ (+w) insertion on the X chromosome. Seven of the fourteen were semi-lethal lines, in which only a few adult males survived. PCR analysis with dtorsin gene-specific primers demonstrated that males from each of the seven semi-lethal lines lacked the dtorsin gene, while males of the seven viable lines retained the dtorsin gene (data not shown). The targeted homologous recombination event was expected to yield the same genomic structure in all correctly targeted lines. To confirm this, we performed Southern blot analysis on two semi-lethal lines, each derived from a different donor line , leaving no potential to produce Dtorsin protein in these lines. Anti-Dtorsin antibody revealed a 38 kDa protein band, the predicted molecular weight of the Dtorsin protein, in wild type adult whole body extracts and head extracts  larvae developed to the adult stage when maintained under the same conditions. In mixed cultures where larvae of both genotypes were present, very few dtorsin null males developed to the adult stage  male larvae moved at 53.3±2.1 strides per minute (n = 33), KO13dtorsin male larvae exhibited approximately a ∼50% decrease in stride frequency, 24.6±3.0   compared to the equivalent dtorsin-null male larvae  lacking the UAS-dtorsin transgene . With the dopaminergic tissue-specific driver TH-GAL4 and the UAS-dtorsin transgene, we observed a significant increase of peristaltic frequency  compared to the control (KO13y w dtorsin), although the rescue was weaker than that achieved with elavGAL4, suggesting that other neurons in addition to dopaminergic neurons may play important roles in regulating larval mobility in dtorsin null mutants. Transgenes of elavGAL4 or TH-GAL4 alone had no effect on the mobility of larvae with or without the KO13dtorsin null mutation . These results suggested that dtorsin expression in neurons plays an important role in regulating larval mobility.To determine whether the larval mobility deficit was specifically due to lack of Dtorsin in neurons, we used the GAL4/UAS system  drivers . When thmutation . Expressdtorsin null mutant mobility defect. We found that human torsinA cDNA expressed with the Actin5c- or the Tubulin-GAL4 drivers rescued all phenotypes  at very low frequency  compared to the control dtorsin null male larvae  . These data indicate that the neuronal functions of the human torsinA and dtorsin are very well conserved with respect to the regulation of the larval locomotion.Next, we examined if the wild type human torsinA cDNA could rescue the requency . When th n = 32) . The perDrosophila, ingestion of dopamine increases dopamine pools in the fly head, though in mammals peripheral dopamine does not enter into the brain dtorsin in dopaminergic cells produced detectable rescue of the locomotion phenotype, we tested whether addition of dopamine to fly food could also rescue this defect. Previous studies have shown that dopamine, serotonin, and octopamine can all affect larval locomotion as well as adult behaviors in Drosophiladtorsin null larvae.In KO13dtorsin mutant larvae  compared to that of the KO13dtorsin larvae grown without drug supplementation . However, no significant changes were detected in the mutant larvae grown with 20 mM octopamine  or 10 mM serotonin  supplementation. No significant differences were observed between wild type larvae after supplementation with dopamine , octopamine , or serotonin  and those grown without monoamine supplementation   .KO13dtorsin male mutant larvae also exhibited relatively straight crawling patterns, similar to those of the wild type larvae with or without dopamine  female larvae contained 0.0631±0.0025 ng dopamine/brain , whereas dtorsin heterozygous female larvae (KO78/+dtorsin) had 0.0340±0.0014 ng dopamine/brain (n = 3) corresponding to a 46% reduction (p<0.001)   . Dopaminp<0.001) .dtorsin mutation. Turnover of dopamine during synaptic release can be inferred by the levels of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) and by the ratio of DOPAC to dopamine in flies, as in mammals dtorsin heterozygous mutant were decreased, the reduction was clearly not proportional to the reduction in dopamine levels. In consequence, the resulting DOPAC:dopamine ratios were elevated over two-fold  is expressed in dopaminergic neurons in the brains of dtorsin null mutants Drosophila larvae and consists of four clusters of 4–10 neurons in each brain hemisphere and a stereotyped pattern of three to five paired or unpaired neurons per segment in the ventral ganglion KO13dtorsin third instar larvae and that the number of dTH-positive cell bodies was approximately normal and clearly not sufficiently reduced to account for the dramatic loss of dopamine observed. However, we did observe some apparent morphological changes in the positioning of cell bodies of dTH-positive neurons and in the patterning of dTH-positive axons in the central brain region of the hemizygous dtorsin null male larvae  catalyses the first and rate-limiting step in the dopamine biosynthesis pathway and is expressed in all dopaminergic neurons. Therefore, we next examined third instar larval brain using anti-dTH antibody to determine whether e larvae . Furtherdtorsin and further pursued a putative role in dopamine trafficking by first conducting gene interaction analysies. We tested for genetic interaction of the null KO13dtorsin allele with mutant alleles of ple , which encodes TH Pu (Punch), the Drosophila ortholog of GTP cyclohydrolase (GTPCH) DAT, encoding the dopamine transporter Pu and ple loss-of-function mutants show similar phenotypes to each other Pu mutants exhibit reductions in dopamine pools due to limiting BH4 levels Pu loss-of-function mutants die during late embryogenesis with completely unpigmented and weak cuticles GCH1 gene, while mutations in the TH gene are responsible for a rare recessive form DAT, was found to be mutated in a hyperactive fly line, fumin. Elimination of DAT activity in the fuminDAT mutant caused a dramatic increase in the length of the waking phase Dopamine synthesis is directly regulated by the specific activity of TH, which, in turn, is modulated by the amount of its cofactor, tetrahydropterin (BH4). In consequence, TH activity is regulated indirectly by the activity of the rate-limiting enzyme in BH4 synthesis, GTP cyclohydrolase (GTPCH) in flies, as in mammals dtorsin null mutation and loss-of-function mutations of Pu, ple, or fuminDAT, we analyzed the peristaltic stride frequency of the double-heterozygous female mutant larvae. No phenotype was seen in any single heterozygous mutant line. The stride frequencies of Z22Pu/+ males  and females  were statistically similar to the stride frequency of the KO13dtorsin/+ females . However, the double heterozygous KO13dtorsin/+; Z22Pu/+ females showed very significantly reduced peristaltic frequency , relative to either single heterozygote , KO13dtorsin/+; 2ple/+ female larvae showed a slight but significant reduction of peristalsis frequency , compared to KO13dtorsin/+; +/+ females  (There was a much weaker interaction between  n = 20) .dtorsin and fuminDAT. The double heterozygous mutant KO13dtorsin/+;fuminDAT/+ females showed similar peristaltic frequency  compared to single heterozygous mutant KO13dtorsin/+ females   that was statistically identical to dtorsin mutant KO13dtorsin/Y males .No statistically significant interaction was detected between , n = 7) . The DATdtorsin null mutation showed a very strong genetic interaction with the Punch (GTPCH) mutation, a weaker interaction with the ple (TH) mutation, and no detectable interaction with the fumin (DAT) mutation.Thus the Pu gene/GTPCH protein activity. To test this hypothesis, extracts were prepared from wild type (y w) and dtorsin heterozygous mutant (KO13y w dtorsin/+ and KO78w dtorsin/+) adult heads and TH and GTPCH activities were compared  and the wild type flies (y w) . However, a significant reduction in GTPCH activity was observed in the dtorsin null mutant heterozygous flies  compared to wild type flies (y w) . These results suggest that the dtorsin is a novel positive-regulator of GTPCH activity in Drosophila.The above findings suggested that the Dtorsin function may regulate the compared . No signdtorsin might be required for maintaining GTPCH protein levels. To test this hypothesis, we prepared extracts from heads of wild type adult males (y w), dtorsin heterozygous adult females (KO13y w dtorsin/+), and dtorsin hemizygous adult males (KO13y w dtorsin/Y) and compared TH and GTPCH protein levels by Western blot analysis (dtorsin null hemizygous flies (KO13y w dtorsin), heterozygous flies , and of wild type flies (y w)  compared to wild type (y w). Moreover, only very low levels of GTPCH protein level were detected in dtorsin null mutant hemizygous males, suggesting only a very small amount of GTPCH proteins are present in the hemizygotes , related to the Clp protease/heat shock family TOR1A is notable for causing early-onset dystonia due to a codon deletion that removes a particular glutamic acid residue from the torsinA protein. TorsinA-null knock-out mice (Tor1a/−−) have been created and die at birth with abnormal nuclear membrane morphology in neural cells dtorsin is most closely related to TOR1Adtorsin revealed that it is an essential gene in flies, as mutants typically fail to reach adulthood and those that do survive fail to reproduce. Null dtorsin mutant flies display both morphological and behavioral phenotypes that may provide starting points for elucidating activities of the Dtorsin protein, using both biochemical and genetic strategies. Our initial examination of these phenotypes indicates that at least one major disruption in dtorsin mutant flies involves regulation of dopamine, a function also implicated in human dystonia dtorsin null mutant will facilitate investigation of this activity and other effects of torsin deficiency that are likely to emerge with more detailed study.The human genome contains four paralogous genes of the torsin family, while dtorsin mutants because of the phenotypes exhibited by rare adult survivors, which had a pale body color and thin bristles  gene and GTPCH is produced from the Pu (Punch) gene. Our finding that genetic interaction of mutations in dtorsin and Pu produces a strong defect in the larval locomotor assay whereas genetic interaction of dtorsin and ple produces a weaker but notable effect , while torsinA is altered in DYT1 dystonia. Deficits in dopamine neurotransmission have been implicated in all these forms of dystonia that have a similar clinical appearance, manifesting as early onset generalized dystonia dtorsin fly lines do not express any functional Dtorsin protein. Thus, the fly mutant is fundamentally different from other DYT1 disease model systems. The interaction between the DYT1-mutant form of the torsinA protein (for example ΔGAG torsinA) and the wild type torsinA protein could be very important for the DYT1 disease manifestations, presumably leading to a decrease in wild type activity. Consequently, it is essential to understand the normal function of the torsinA protein in order to understand how compromise of its function leads to DYT1 dystonia. The availability of fly lines mutant for dtorsin provides the basis for applying the powerful tools of Drosophila melanogaster genetics to define the function(s) of the sole torsin family member in this species, with the expectation that these activities may provide insight into torsinA function in humans. In this regard, the ability of the human torsinA cDNA to rescue the mobility defect of the dtorsin null mutant demonstrates that at least the torsin function involved in regulation of larval locomotion, which requires expression in neurons, is well conserved between the fly and the human. The dtorsin null fly system will therefore permit analysis of the effects of various DYT1-causing mutations on this function, using the biochemical, pharmacological, and genetic approaches.It is important to stress that the mutated abnormal torsinA protein is expressed together with normal torsinA in the DYT1 animal models and DYT1 patients, while the null y w; P{70FLP}11 P{70I-SceI}2B Sco/CyO, 2S line for the gene targeting, GAL4 driver lines, and the FM7i, P{w[+mC] = ActGFP}JMR3/C(1)DX, y f were obtained from the Bloomington Stock Center. The MHC-GAL4 line was obtained from Dr. Aaron DiAntonio . The fuminDAT line was a kind gift from Dr. F. Rob Jackson .Flies were kept on standard medium, except for larval locomotion analysis (see below). The dtorsin was amplified from genomic DNA  by PCR using the following primers: 5′- AGCGGCCGCGATATTATGACCCTGGCCAAGGGCTACTTGGAGCAGCTACCAGGACCGGGCAAGGCATCCTCCTATCGCT -3′ (TK4A) and 5′-CGGTACCGCAAGGAAAATTCCGCGGTGGAATGTATAGATGGGCAAAAGCGATAGGCAGCATAAGACTAGA-3′ (TK3A). The product was digested with NotI and Acc65I and cloned into the NotI and Acc65I sites of pw25.The “ends-out” targeting scheme was a modified version of a method described previously dtorsin was amplified from genomic DNA by PCR using the following primers: 5′-AGGCGCGCCGGCCATGGCCATTTACTGAGAAAGGACCGACCGACTCCACATTTTAATGC -3′ (TK2) and 5′-CCGTACGCAATGCTGTATATATTAATCGTTTGAGTATAAACAAATATCACGACTTA-3′ (TK1). The product was digested with AscI and BsiWI and cloned into the AscI and BsiWI sites of the plasmid made in step 1. Nine fly lines carrying independent insertions of the donor construct were generated using standard procedures. Two independent lines, B2-1 (2nd chromosome insertion) and B7-1 (3rd chromosome insertion) were used for further experiments. Males heterozygous for each donor insertion were crossed to 2 CyOy w; P[70FLP]11 P[70I-SceI]2B Sco/S females, and resulting larvae were heat-shocked at 38°C for 1 hour at 3 days after egg laying (AEL). The resulting unbalanced females were mated in groups of 10 to 2 CyOy w; P[70FLP]11 P[70I-SceI]2B Sco/S males. The progeny from this cross were heat-shocked at 3 days AEL, and 405 vials were screened for red eye female upon eclosion. Non-mosaic, unbalanced flies were saved as FLP-insensitive integrations. In total 14 FLP-insensitive integrations were found that mapped to the X chromosome. Of those, seven lines were hemizygous viable and seven  were semi-lethal. Few male adults survived from the seven semi-lethal lines, and no homozygous females survived.In step 2, a 3.5 kb genomic fragment of the 3′ portion of KO13y w dtorsin/Y and KO78w dtorsin/Y.Southern blots were performed using standard techniques. The DNA was prepared from adult male survivors of dtorsin coding sequence by PCR for radio labeled probes (Probe 1 and Probe 2) (5′- GGATCCACCCGTGGAGCCGGCGAAACTTTTCCGGGTGCAG -3′ and 5′- GGCCATGGCCATTTACTGAGAAAGGACCGACCGACTCCAC -3′ and detected a 1.5 kb BamHI fragment in wild type (y w and w) in genomic Southern blots (5′- GATTGCAGCACTTGCCGGCAACCGTTTCTATCGATAGTTG -3′ and 5′- GGATCCTGGACAGGATACGCTTGACGGATGGGCCTTGTTG -3′ and detected a 0.5 kb BamHI fragment in wild type (y w and w) (Two DNA fragments were generated to the 5′ and 3′ adjacent region of Probe 2)  and gel rn blots . A 381 bw and w) .dtorsin was amplified from genomic DNA by PCR using the following primers: 5′- GCGGCCGCTTGGACCTGGACTTTTCGGGATCCTGGACAGGATACG -3′ and 5′-GAATTCCCTCAAAGATCTGGGACTAGACGGAAAGCAGAGCGATTT-3′. The fragment contains 0.4 kb 5′ non-coding region, the whole coding region of the dtorsin gene, and 0.37 kb 3′ non-coding region. The product was digested with EcoRI and NotI and cloned into the EcoRI and NotI sites of pCaSpeR3. Four fly lines (GDT101-1∼4) carrying independent insertions of the donor construct were generated using standard techniques. All four lines were crossed to individual dtorsin mutant lines.A 1.9 kb genomic fragment containing dtorsin cDNA was amplified from LD20854  by PCR using the following primers: 5′-CCGGAATTCCCTTGCATTTTATGATGAGCTTTCCACGCATGTTA-3′ and 5′-GTTGCGGCCGCCATTAAAATGTGGAGTCGGTCGGTCCTTTCTCA-3′. The product was digested with EcoRI and NotI and cloned into the EcoRI and NotI sites of pUAST A 1.2 kb -dtorsin transgene on the second chromosome was used for the rescue experiments.The transgenic line B5 with UAS5′-GCGGGATCCATTCATGAAGCTGGGCCGGGCCGTGCTGGGCCTGC-3′ and 5′-CTCGAGCGGCCGCTCAATCATCGTAGTAATAATCTAACTTGGTG-3′. The product was digested with Acc65I and NotI and cloned into the Acc65I and NotI sites of pUAST.A 1.0 kb human torsinA cDNA was amplified from pcDNA3-htorA The transgenic line #8 with UAS-htorA transgene on the second chromosome was used for the rescue experiments.Brains of third instar larvae or heads of 48–72 hr post-eclosion adult flies were homogenized in 0.1 M perchloric acid. Monoamines were separated by HPLC analysis using a CoulArray HPLC system  and a Synergi 4 µm Hydro-RP column  as described The specific activity of tyrosine hydroxylase (TH) in tissue extracts was determined as described previously Approximately 50 heads from 48–72 h post-eclosion adults were homogenized in 100 µl of cold 50 mM Tris, pH 8.0 solution containing 2.5 mM EDTA and 5% sucrose. After centrifugation of the homogenates at 9,300 x g for 10 minutes at 4°C, 5 mg of activated charcoal was added to 100 µl supernatant with gentle mixing to absorb eye pigment. Samples were centrifuged to remove the charcoal, and protein concentrations were determined using BioRad protein assay reagent. Forty-five µg of protein from tissue extracts was mixed with GTP to a final concentration of 0.25 mM in a final volume of 70 µl. After incubation at 37°C for 1 hour, the product of the reaction, 7,8-dihydroneopterin triphosphate, was oxidized to its fluorescent form in 30 µl of 1% iodine and 2% potassium iodide in 1 mM HCl, and incubated for 1 hour at room temperature. The samples were then centrifuged at 14,500 x g for 5 minutes at room temperature. The oxidation reaction was terminated and the samples decolorized with the addition of 15 l of 3% ascorbic acid. The samples were adjusted to pH 8.0, and 50 µl of each sample was mixed with 2 µl of calf intestine alkaline phosphatase, 7 µl of alkaline phosphatase buffer  and 11 µl of distilled water. The mixtures were incubated at 37°C for 30 minutes to dephosphorylate the neopterin triphosphate product. The supernatant was filtered and then separated by HPLC as described above, except that neopterin was detected by fluorescence at excitation 360 nm/emission 465 nm, and its content was determined by comparison to commercial neopterin (Sigma-Aldrich) as standard using ESA CoulArray software.y w or KO13y w dtorsin) were homogenized in 300 µl of RIPA buffer ), and the homogenate was sonicated and then clarified by a brief centrifugation . For detection of protein in adult heads, heads were removed manually from 50–60 flies of each genotype and were homogenized either in 100 µl phosphate-buffered saline (PBS) with Protein Inhibitor Cocktail  or in 100 µl RIPA buffer, as above. The protein concentrations were determined with the Dc Protein Assay Reagent . The proteins (20–30 µg) were separated by electrophoresis in 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk in PBS with 0.1% Tween 20 before incubation with primary antibodies. Rabbit anti-Dtorsin antibody raised against amino acids 51–339 of Dtorsin was a gift of Dr. Vijaya Ramesh  and used at 1∶2000 dilution. GTPCH was detected using affinity-purified polyclonal anti-GTPCH isoform B/C antibody Drosophila TH For analysis of whole flies, thirty adult male flies  in 7 mls of water.The larval locomotion assay was carried out at room temperature. Wandering third instar larvae of a particular genotype were picked from the vials. Male larvae were distinguished from females by the presence of larger genital discs as described previously Larval locomotion was recorded for 60 seconds using a Canon Powershot G7 attached to a Nikon SMZ800 microscope. Peristalsis frequency was counted manually from the QuickTime movie.Larval movement was also recorded at 10 frames per second for 60 seconds using a digital video camera recorder . Video tracking was performed using Prairie Dog, an application to trace the movement of an object in a series of TIFF files. Prairie Dog is a new application specifically developed by us to analyze the larval movement and is available upon request from H. M. Some locomotion analysis employed ICARUS software (Tracking/New User Feature) y w) and KO13y w dtorsin were transferred individually to 1.0 g FORMULA4-24 instant Drosophila media in 5 ml water or 5 ml of 20 mM dopamine-hydrochloride (Sigma-Aldrich), 20 mM octopamine hydrochloride (Sigma-Aldrich), or 10 mM serotonin hydrochloride (Sigma-Aldrich). Larval locomotion assays were performed, as described above.First instar larvae of the wild type  and BamHI sites (B) are indicated. B. Southern blot of BamHI digested genomic DNA, confirming proper integration of the targeting construct. Probe 1 detected 1.5 kb BamHI fragment in the BamHI digested genomic DNA of the wild type (y w/Y and w/Y) males and 5.7 kb BanHI fragment in KO13y w dtorsin/Y and KO78w dtorsin/Y males. Probe 2 detected 0.5 kb BamHI fragment in the BamHI-digested genomic DNA of the wild type (y w/Y and w/Y) males and 5.7 kb BanHI fragment in KO13y w dtorsin/Y and KO78w dtorsin/Y males.(TIF)Click here for additional data file.Figure S2Dtorsin immunoreactivity is reduced in dtorsin hemizygous adult extracts.A. Anti-Dtorsin antibody recognizes a 38 kDa protein band in the wild type (y w) adult whole body (lane 1) and adult head (lane 3) extracts that is absent in dtorsin-null (KO13y w dtorsin) whole body (lane 2) and head (lane 4) extracts. Thirty µg of proteins were loaded in each lane. B. Anti-Actin antibody recognize a 42-kDa protein (Drosophila Actin) that are present in a similar amount both in the wild type (y w) adult whole body (lane 1) and adult head (lane 3) extracts as well as in dtorsin-null whole body (lane 2) and head (lane 4) extracts. Thirty µg of proteins were loaded in each lane.(TIF)Click here for additional data file.Figure S3dtorsin loss-of-function mutant flies exhibit pigmentation and bristle phenotypes.A–C, Pigmentation and bristle phenotypes of dtorsin mutants. Adult males of the genotype y w/Y (A), KO13y w dtorsin/Y (B), KO13y w dtorsin/Y; GDT101-2 (C). dtorsin null male KO13dtorsin (B) had shorter, thinner bristles with reduced pigmentation in the thorax compared to wild type (A). Both larger bristles (macrochaetae) and smaller bristles (microchaetae) were affected. These bristle phenotypes were completely rescued by the introduction of 1.9 kb genomic fragment containing the entire dtorsin gene (GDT101) (C).(TIF)Click here for additional data file.Figure S4Effect of dopamine feeding on the adult pigmentation.A–D, Effect of dopamine on the pigmentation phenotype of dtorsin mutants. Adult males of the genotype y w (A), y w+50 mM dopamine (B), KO13y w dtorsin (C), and KO13y w dtorsin+50 mM dopamine (D) flies. The pictures for A–D were taken one day after eclosion to ensure pigmentation reached the maximum level. Dopamine was added to the fly food during the larval period, as described in (TIF)Click here for additional data file.Figure S5The double heterozygous combinations for dtorsin and DAT mutants have similar mobility compared to the wild type larvae. Peristaltic frequencies for the wandering stage third instar larvae of the genotype KO13dtorsin/+; female (n = 7), and KO13dtorsin/+; fuminDAT/+ female (n = 14) are shown. Results are the mean ± S.E.M. No significant difference between the control females (KO13dtorsin/+) and the single heterozygous females .(TIF)Click here for additional data file.Video S1y w) third instar larva for 60 seconds. Wild type larvae showed smooth peristaltic movement of 40 to 60 strides per minute.Shown is the crawling behavior of wild type ((MP4)Click here for additional data file.Video S2dtorsin mutant male (KO13y w dtorsin/Y) third instar larva. dtorsin mutant larvae stopped more frequently and changed directions of movement more often than the wild type.Shown is the crawling behavior of (MP4)Click here for additional data file.Table S1(DOCX)Click here for additional data file.