Introduction {#S1}
============

IFN-γ is primarily produced by innate cells, including natural killer (NK) and natural killer T (NKT) cells, non-cytotoxic innate lymphoid cells ^[@R1]^, and adaptive immune cells, including CD4^+^ T helper (Th) 1 and CD8^+^ cytotoxic T lymphocytes (CTL) cells. ^[@R2]^ IFN-γ activates macrophages to produce pro-inflammatory cytokines^[@R3]^, but can also counteract inflammatory and immunostimulatory pathways^[@R4]^. Transgenic mice engineered to express IFN-γ in airway epithelial cells were found to present with increased inflammation and emphysema^[@R5]^ but also with reduced eosinophilia and airway reactivity^[@R6]^. However, because airway epithelial cells do not express IFN-γ the findings in these transgenic mice may not be physiologically relevant.

For clinical applications, IFN-γ has beneficial effects when administered as prophylactic treatment of lupus nephritis^[@R7]^ and in cases of rheumatoid arthritis.^[@R8]^ IFN-γ signaling inhibits neutrophil accumulation in the lung^[@R9]^ or suppresses neutrophil production in the bone marrow.^[@R10]^ These anti-inflammatory responses are mediated by induction of cytokine decoy receptors, IL-1Rα and IL-18BP.^[@R2]^ Further, IFN-γ tempers the development of erythrocytes and eosinophilic granulocytes ^[@R11]^, and inhibits Th2 cell function. ^[@R12]^

Because chronic inflammation promotes chronic lung diseases^[@R13]^, identifying the intracellular factors that mediate the anti-inflammatory action of IFN-γ can be of great importance. Our previous studies have shown that IFN-γ causes cell death in airway epithelial cells by activating STAT1^[@R14]^, inducing expression of the BH3-only protein, Bik^[@R15]^, and suppression of Bmf expression to induce autophagy.^[@R16]^ Bik and Bmf are Bcl-2 family members that share only the Bcl-2 homology region 3 (BH3) such as Noxa, Puma, Bim, Bid, Bad, Bid, Bnip-3L, and Hrk. This group of proteins is believed to sense cell death signals that emanate from internal stresses such as DNA damage^[@R17]^ or CAP-independent translation^[@R18]^ or from external stimuli, such as IFN-γ^[@R15]^ or other cytokines.^[@R19]^

We found that in airway epithelial cells IFN-γ dramatically induced the BH3-only protein, Noxa, but that Noxa did not induce cell death, yet inhibited inflammation in response to allergen. Noxa required the interaction with phosphorylated HSP27 via a covalent disulfide bond to suppress the nuclear translocation and activation of NF-κB by inhibiting degradation of ubiquitylated κBa. Furthermore, inducible expression of Noxa in airway epithelia of adult mice suppressed allergen-induced inflammation *in vivo*. However, the cross-linking Cys25 was not necessary when the N-terminal peptide was used to block IκBα degradation and inflammation. Therefore, these studies identify the anti-inflammatory mediator of IFN-γ to be Noxa cross-linked to the non-Bcl-2 protein, pHSP27 that renders N-terminal region of Noxa functional.

Results {#S2}
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IFN-γ rapidly induces Noxa expression to suppress inflammation {#S3}
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Noxa mRNA levels were dramatically induced by 25-50 ng/ml IFN-γ treatment over 24-48 h in primary HAECs from non-diseased individuals ([Fig. S1A](#SD1){ref-type="supplementary-material"}). Induction occurred within 1 h of IFN-γ treatment both for Noxa mRNA ([Fig. 1A](#F1){ref-type="fig"}), and protein levels ([Fig. 1B](#F1){ref-type="fig"}), while Hrk, Bad, or Puma showed no change in expression (data not shown). This rapid induction by IFN-γ was consistently observed in HAECs from 14 individuals ([Fig. 1C](#F1){ref-type="fig"}). As expected, Noxa protein and mRNA were undetectable in MAECs from *noxa*^-/-^ mice but Noxa expression was increased by IFN-γ in wild-type MAECs ([Fig. S1B](#SD1){ref-type="supplementary-material"}). IFN-γ failed to upregulate Noxa expression in MAECs from *STAT1^-/-^* mice ([Fig. S1B](#SD1){ref-type="supplementary-material"}). However, unlike the DNA damage-induced Noxa expression ^[@R20]^, IFN-γ-induced Noxa expression was independent of p53 ([Fig. S1C](#SD1){ref-type="supplementary-material"}).

Noxa deficiency causes increased inflammation {#S4}
---------------------------------------------

While Noxa mRNA and protein levels were induced robustly by IFN-γ, *noxa*^+/+^ and *noxa*^-/-^ airway cells were both susceptible to IFN-γ-induced cell death ([Fig. S1D](#SD1){ref-type="supplementary-material"}). However, we observed that *noxa*^-/-^ compared with *noxa*^+/+^ mice developed increased inflammation following intranasal instillation with house dust mite allergen for 5 consecutive days. The numbers of total cells ([Fig. 1D](#F1){ref-type="fig"}) eosinophils and macrophage, but not neutrophils and lymphocytes ([Fig. 1E](#F1){ref-type="fig"}) recovered by bronchoalveolar lavage (BAL) were significantly higher in *noxa*^-/-^ compared with *noxa*^+/+^ mice. In addition, when sensitized with ovalbumin (OVA) injection on days 1 and 7 and challenged with OVA aerosols for 5 d, mucous cell numbers were higher ([Fig. S2A](#SD1){ref-type="supplementary-material"}) and inflammatory cells were more evident in *noxa*^−/−^ compared with *noxa*^+/+^ mice ([Fig. S2B](#SD1){ref-type="supplementary-material"}). These observations suggested that Noxa may play a role in blocking allergen-induced inflammation, but not in the cell death process of hyperplastic airway epithelial cells. Previous studies have shown that Noxa expression sensitizes cells to apoptosis ^[@R21]^. To determine whether Noxa sensitizes airway epithelial cells, we first treated HAECs with increasing H~2~O~2~ concentrations and found that 45 μM affects cell viability slightly ([Fig. S2C](#SD1){ref-type="supplementary-material"}). However, when combined with increasing multiplicity of infection (MOI) of Ad-Noxa, cell viability was progressively reduced compared to the Ad-GFP controls ([Fig. S2D](#SD1){ref-type="supplementary-material"}).

Because expression of many pro-inflammatory genes are regulated by NF-κB including IL-8,^[@R22]^ we investigated whether Noxa suppresses the synthesis of inflammatory chemokines. The TNFα-induced IL-8 secretion from human airway epithelial cells was significantly reduced when Noxa was expressed using an adenoviral expression vector (Ad-Noxa) compared to Ad-GFP-infected controls ([Fig. 2A](#F2){ref-type="fig"}). The possible role of Noxa in affecting NF-κB activation was tested using the A549-NF-κB-luc cells that are stably transfected with a luciferase reporter gene driven by a promoter containing several NF-κB response elements. As expected, TNFα-induced luciferase activity was inhibited when Noxa was expressed in a dose-dependent manner ([Fig. 2B](#F2){ref-type="fig"}); no cell death was detected by Ad-Noxa even at a MOI of 300 (data not shown). Further, suppression of Noxa expression using shRNA enhanced IFN-γ-induced NF-κB activity in cells treated with TNFα for 30 min ([Fig. 2C](#F2){ref-type="fig"}). Further, merely expressing Noxa or expressing Noxa in the presence of IFN-γ treatment showed a similar reduction in A549-NF-κB-luc cells, showing that IFN-γ had no further impact on Noxa-mediated suppression of NF-κB activity ([Fig. S2F](#SD1){ref-type="supplementary-material"}). Together, these findings suggest that the anti-inflammatory effect of IFN-γ is mediated by Noxa. We next analyzed the effects of IFN-y or Noxa on TNFα-induced change in sub-cellular localization of NF-κB. While NF-κB was localized to the nucleus 30 min after TNFα treatment, it remained localized to the cytosol in cells that were pretreated with IFN-γ for 24 h ([Fig. 2D](#F2){ref-type="fig"}). Similarly, direct expression of Noxa using adenoviral vectors suppressed TNFα-induced nuclear translocation of NF-κB as shown by immunofluorescence ([Fig. 2E](#F2){ref-type="fig"}). In addition, IFN-γ was delayed in suppressing nuclear translocation of NF-κB in *noxa^-/-^* MAECs ([Fig. 2F](#F2){ref-type="fig"}), confirming that IFN-γ requires Noxa to delay NF-κB activation. These findings were further confirmed by immunoblotting of nuclear and cytoplasmic extracts that showed nuclear NF-κB being significantly reduced in cells pretreated with IFN-γ compared to non-treated controls ([Fig. S2F](#SD1){ref-type="supplementary-material"}).

Noxa blocks the degradation of ubiquitylated IκB-α {#S5}
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In non-stimulated cells, the inhibitory IκBα protein sequesters NF-κB to the cytoplasm and inflammatory stimuli activate IKKα and IKKβ to phosphorylate IκBα, which leads to its ubiquitylation and proteasomal degradation, releasing NF-κB to translocate to the nucleus.^[@R23],\ [@R24]^ Over 0, 15, and 30 min of TNFα treatment, IκBα levels were reduced in the cytosolic extracts of *noxa^-/-^* but not *noxa*^+/+^ MAECs ([Fig. S2G](#SD1){ref-type="supplementary-material"}), suggesting that Noxa stabilizes IκBα. TNFα-induced nuclear translocation of NF-κB was also significantly reduced following overexpression of Noxa using Ad-Noxa compared to cells infected with Ad-GFP ([Fig. 2G](#F2){ref-type="fig"}), suggesting that Noxa expression is sufficient to stabilize IκBα. Interestingly, after 10 min of TNF-α treatment the levels of phosphorylated IκBα were similar in HAECs infected with Ad-Noxa and Ad-GFP as control ([Fig. 2H](#F2){ref-type="fig"}). Lysine-48 (K48)-linked polyubiquitin chains are well established as the canonical signal for proteasomal degradation ^[@R25]^. Interestingly, the levels of IκBα with homotypic K48-linked ubiquitin chains were increased in HAECs infected with Ad-Noxa and expressed Noxa compared with Ad-GFP-infected controls ([Fig. 2I](#F2){ref-type="fig"}). This finding suggests that Noxa blocks the proteasomal degradation of IκBα that is already destined for degradation.

Noxa is cross-linked to phosphorylated HSP27 {#S6}
--------------------------------------------

Because Noxa is known to inactivate Mcl-1 and thereby sensitize cells to apoptotic death^[@R26],\ [@R27]^ we tested the hypothesis that Noxa may interact with other protein(s) to gain anti-inflammatory properties. Noxa antibodies consistently detected a cross-reacting protein of approximately 32 kDa in size only in IFN-γ-treated HAECs ([Fig. 3A](#F3){ref-type="fig"}). Peptide mass fingerprint analysis of the cross-reacting the 32 kDa band excised following immunoprecipitation repeatedly revealed a high score for HSP27 ([Table S1](#SD1){ref-type="supplementary-material"}). This observation was confirmed by immunoblotting of anti-Noxa immunoprecipitated proteins with an antibody to HSP27 phosphorylated at Ser~82~ ([Fig. 3B](#F3){ref-type="fig"}). Interestingly, Mcl-1 and Bcl-x~L~, proteins known to interact with Noxa, ^[@R27]^ were not detected in the immunoprecipitates ([Fig. 3B](#F3){ref-type="fig"}). Immunoprecipitation with p-S~82~HSP27 antibodies confirmed that Noxa forms a complex with phosphorylated HSP27 (pHSP27) ([Fig. 3C](#F3){ref-type="fig"}). The interaction between Noxa and pHSP27 was further verified by pull-down assays from extracts prepared from AALEB cells using recombinant, purified GST-Noxa protein and detecting pHSP27 protein by Western blot analysis ([Fig. 3D](#F3){ref-type="fig"}). Blocking the phosphorylation of expressed HSP27 using the p38MAPK inhibitor, SB203580, reduced the levels of immunoprecipitated pHSP27-Noxa complex ([Fig. 3E](#F3){ref-type="fig"}), further supporting that pHSP27 rather than total HSP27 needs to be present to form the Noxa/pHSP27 interaction. Interestingly, merely overexpressing Noxa by infecting cells with Ad-Noxa in the absence of IFN-γ also resulted in the appearance of the 32 kDa Noxa-pHSP27 complex ([Fig. S3A](#SD1){ref-type="supplementary-material"}), suggesting that IFN-γ was not inducing a cross linking enzyme to facilitate this complex formation. The idea that Noxa and p-HSP27 are linked by disulfide bond(s) was confirmed by the absence of the p-HSP27-GST-Noxa complex in pull-down products from cell extracts treated with the reducing reagent, DTT that cleaves disulfide bonds ([Fig. S3B](#SD1){ref-type="supplementary-material"}).

To identify the amino acids within Noxa that are responsible for the Noxa-HSP27 complex, we generated various mutant GST-Noxa proteins that were tested in pull-down assays. Deletions of 10 amino acids from the N-terminus (Noxa-Δ5′) or 14 amino acids from the C-terminus (Noxa-Δ3′), abrogated the interactions of Noxa and pHSP27 ([Fig. S3C](#SD1){ref-type="supplementary-material"}). Because 293T cells express no detectable levels of endogenous HSP27, these cells were co-transfected with an HSP27-expression vector along with wild-type Noxa, Noxa-2CS, and Noxa-3KR. Mutations of cysteines 25 and 51 to serines (Noxa-2CS) also disrupted the interactions while mutation of lysines 35, 38, and 41 to argnines (Noxa-3KR) did not ([Fig. 3F](#F3){ref-type="fig"}). Further mutagenesis experiments demonstrated that only Cys~25~ residue was essential for the formation of Noxa-phospho-HSP27 complex ([Fig. 3F](#F3){ref-type="fig"}) confirming the hypothesis that Noxa and HSP27 are linked by an intermolecular disulfide bond.

Because HSP27 contains only one cysteine residue at position 137, the role of this residue was investigated by comparing wild-type and C137A mutant HSP27 constructs expressed in 293T cells that were treated with IFN-γ to induce Noxa. Although expression levels of pHSP27 were equal in cells transfected with either construct, immunoprecipitation with Noxa antibodies showed that the formation of Noxa-pHSP27 was impaired in cells transfected with the C137A mutant and was accompanied by the loss of interactions with IκBα/NF-κB complex ([Fig. 3G](#F3){ref-type="fig"}). These studies demonstrated that an intermolecular disulfide bond between Cys~25~ in Noxa and Cys~137~ in HSP27 cross-links Noxa to pHSP27 to allow the formation of a complex with IκBα and NF-κB.

HSP27 is essential for Noxa-κBα-NF-κB interaction {#S7}
-------------------------------------------------

Suppression of HSP27 in AALEB cells by stably expressing shHSP27 did not affect IFN-γ-induced Noxa expression ([Fig. S3D](#SD1){ref-type="supplementary-material"}). These cells were used to investigate the contribution of pHSP27 in blocking nuclear translocation of NF-κB. As shown by immunofluorescence staining IFN-γ blocked nuclear translocation of NF-κB in shCtrl but not in shHSP27 cells ([Fig. 3H](#F3){ref-type="fig"}). In addition, when Noxa or HSP27 expression in AALEB cells was reduced using shNoxa or shHSP27, IFN-γ no longer suppressed secretion of IL-8 ([Fig. S3E](#SD1){ref-type="supplementary-material"}) or IL-6 ([Fig. S3F](#SD1){ref-type="supplementary-material"}). Therefore, we proceeded to determine whether the interaction of Noxa with IκBα/NF-κB would be disrupted by reducing HSP27 levels. Although NF-κB and κBα were equally expressed in shCtrl and shHSP27 cells, the interactions between Noxa and NF-κB or κBα were abolished in shHSP27 cells ([Fig. 3I](#F3){ref-type="fig"}). Immunoprecipitation with NF-κB (p65) antibodies from shHSP27 cell extracts showed that the interaction of NF-κB with IκBα was essentially completely disrupted when cells were treated with TNFα, suggesting that IκBα degradation occurred faster when HSP27 was suppressed ([Fig. 3I](#F3){ref-type="fig"}). Further, the NF-κB---Noxa interaction was disrupted in shHSP27 cells but not in shCtrl cells ([Fig. 3I](#F3){ref-type="fig"}) and TNFα-induced degradation of IκBα was enhanced in shHSP27 compared with shCtrl cells ([Fig. 3J](#F3){ref-type="fig"}), suggesting that pHSP27 also is crucial in stabilizing IκBα. Therefore, Noxa requires the presence of pHSP27 to interact with the IκBα/NF-κB complex and the cross-linking of pHSP27 and Noxa appears to play a critical role in stabilizing IκBα.

Inducible Expression of Noxa in AECs protects from allergic inflammation and mucous cell hyperplasia {#S8}
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Constitutive expression of HSP27 has been detected in the lungs of mice ^[@R28]^ but the presence phosphorylated HSP27 in various tissues and cell types have not been studied. Because the cross-linking of Noxa to pHSP27 may be specific to airway epithelia, we compared pHSP27 levels in various tissues and cell types. Phospho-HSP27 was detected in MAECs and in lung tissues but not in murine embryo fibroblasts, thymus, and liver tissues ([Fig. 4A](#F4){ref-type="fig"}). To investigate the anti-inflammatory and possibly the therapeutic role of Noxa expression in airway epithelial cells *in vivo*, we generated a conditional inducible transgenic (Noxa^Ind^) mouse using the tetO-CCSP promoter system. Immunofluorescence analyses showed induced levels of Noxa in the bronchial epithelial cells of three founder Noxa^Ind^ mouse lines compared with wild-type littermates ([Fig. 4B](#F4){ref-type="fig"}). These mice expressed Noxa mRNA and protein in the lung tissues 24 h post doxycycline (dox)-diet ([Fig. 4C](#F4){ref-type="fig"}). These transgenic mice and littermate controls were instilled with house dust mite (HDM) extract for 5 consecutive days, given the dox-diet on days 6 and 7, and euthanized on day 8. The number of total inflammatory cells in the BAL fluid was reduced in Noxa^Ind^ mice compared to littermate controls and these reduced numbers were due to reduced BAL eosinophils and lymphocytes ([Fig. 4D](#F4){ref-type="fig"}). The no dox, no HDM, dox-fed but not HDM mice controls showed only macrophages and no inflammation (data not shown). Transgenic expression of Noxa also resulted in a 3-fold reduced number of mucous cells per mm basal lamina (BL) compared with littermate controls ([Fig. 4E](#F4){ref-type="fig"}). Immunofluorescence analysis showed that expressed Noxa was co-localized with NF-κB in the cytosol resulting in the lower percentage of airway epithelial cells with nuclear NF-κB in Noxa^Ind^ mice ([Fig. 4F](#F4){ref-type="fig"}). Further, pHSP27 was co-localized with induced Noxa ([Fig. S4A](#SD1){ref-type="supplementary-material"}) and NF-κB ([Fig. S4B](#SD1){ref-type="supplementary-material"}) in airway epithelial cells of Noxa^Ind^ mice.

The N-terminal Noxa peptide stabilizes IκBα and is anti-inflammatory {#S9}
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We further tested whether specific regions of the Noxa protein may act as anti-inflammatory by using Noxa-derived peptides. Based on our findings that Cys25 constitutes the cross-linking amino acid, and because the human and murine Noxa proteins share a high homology, except that the murine Noxa contains the duplicate sequence ([Fig. S5A](#SD1){ref-type="supplementary-material"}), we selected four regions that are 15 amino acids in length and span the following functional regions of the human Noxa protein. Peptide NoxaA represents the N-terminus, NoxaB contains Cys25, NoxaC comprises the BH3 domain, and NoxaD represents the C-terminal end of the protein ([Fig. S5B](#SD1){ref-type="supplementary-material"}). To facilitate uptake into cells, the peptides were synthesized with the HIV-derived TAT sequence on their N-termini. When A549 cells were treated with 10 ng/ml TNFα, NF-κB-induced luciferase activity, was reduced by NoxaA and NoxaD peptides but not by NoxaB or NoxaC ([Fig. 5A](#F5){ref-type="fig"}). However, when nuclear localization of NF-κB was investigated in TNFα-treated MAECs by immunofluorsence, only NoxaA but not NoxaD or the control TAT peptide affected nuclear localization ([Fig. 5B](#F5){ref-type="fig"}).

To examine the anti-inflammatory effect *in vivo*, *noxa^-/-^* mice were intranasally instilled with 50 μg house dust mite allergen for 5 consecutive days and then instilled with NoxaA peptide on days 6 and 7. The number of inflammatory cells in the BAL fluid mice was significantly reduced by NoxaA ([Fig. 5C](#F5){ref-type="fig"}) and this reduction was due to reduced numbers of eosinophils ([Fig. 5D](#F5){ref-type="fig"}). The number of neutrophils, macrophages, and lymphocytes were not affected (data not shown). After 10 min of TNF-α treatment the levels of IκBα with homotypic K48-linked ubiquitin chains were increased in AALEB cells treated with NoxaA compared to Ctrl peptide ([Fig. 5E](#F5){ref-type="fig"}).

Discussion {#S10}
==========

The present studies show that IFN-γ through STAT1 activation rapidly induces Noxa which is cross-linked with a non-Bcl-2 family member, pHSP27, and suppresses the canonical NF-κB signaling pathway. Therefore, these findings define the mediator by which IFN-γ acts as anti-inflammatory and are the first to show that a Bcl-2 family member could inhibit inflammation by reducing NF-κB activation.

Members of the extrinsic apoptotic machinery, including the TNFR1 interactors, TRADD, FADD, and RIPK1 ^[@R29]^ are known to be required for optimal NF-κB activation. The present study adds a new paradigm by showing that a member of the intrinsic apoptotic machinery affects the immune response by directly blocking NF-κB activation. It is well-established that proteins of the Bcl-2 family interact with each other to control the permeabilization of the mitochondrial outer membrane (MOM) and thereby regulate apoptosis. However, only a limited number of studies have explored their interaction with non-Bcl-2 family members and their roles in cellular processes other than those related to cell death. For example, Bad effects insulin secretion and metabolism ^[@R30]^ and BID, contrary to Noxa, by interacting with NOD1, NOD2, and the IκB kinase (IKK) complex facilitates NF-κB activation.^[@R31]^

Noxa is induced rapidly also by hypoxia via hypoxia-inducible factor (HIF)-1α^[@R32]^ by DNA damage,^[@R17]^ by ER stress inducing agents.^[@R33]^ Furthermore, anticancer agents, including the proteasome inhibitor, bortezomib, or ubiquitylation of Mcl-1, induce Noxa expression.^[@R35]^ All these studies used fibroblasts, chronic lymphocytic leukemia, melanoma, neuroblastoma, osteosarcoma, and lung cancer cells and investigated expressed Noxa interacting with the anti-apoptotic Mcl-1 to regulate cell death by affecting Mcl-1 degradation.^[@R25],[@R36]^ However, although Mcl-1 was present in airway epithelial cells, the presence of pHSP27 caused the IFN-γ-induced Noxa to prefer the cross-linking with pHSP27 and not Mcl-1. Ser~13~ phosphorylation alters Noxa structure and blocks the interaction site with Mcl-1.^[@R34]^ Whether pHSP27 phosphorylates Noxa or just forces the N-terminal region to be exposed for interaction with IκBα is unclear. While the phosphorylation and ubiquitylation process remained unaffected, Noxa delayed the degradation IκBα after it was already tagged by the K48 ubiquitin chain. Small molecules, called ubistatins, block the binding of ubiquitylated substrates to the proteasome by targeting the ubiquitin-ubiquitin interface of K48-linked chains.^[@R35]^ Whether the N-terminus of Noxa interacts with the IκBα-specific proteins or the ubiquitin-ubiquitin interface or blocks the interaction of ubiquitylated IκBα to the 19S proteasome before being shuttled for degradation needs further investigation.

The airway epithelium is constantly exposed to the elements of the outer world and therefore has to continuously regulate inflammation. We show that Noxa is rapidly induced within hours and this rapid response allows the fine-tuning or unnecessary enhancement of inflammation. Given that Noxa expression in airway epithelium causes a significant difference in inflammation reveals that this protein contributes to the anti-inflammatory protective role of the airway epithelium. Because many pathways that are not necessarily associated with inflammation, including autophagy, regulators of ROS also play a role in regulating inflammation the induction of Noxa may be one of many mechanisms by which the airway epithelium dampens inflammation. Noxa expression can either sensitize airway cells when cells are damaged or inhibit inflammation. The present studies also suggest that the underlying mechanisms of Noxa-induced cell death in airway epithelial cells need further investigation, because Noxa-induced cell death may not only depend on enhanced degradation of Mcl-1 but also on blocking NF-κB activation that directly regulates expression of the anti-apoptotic Bcl-2.

Both the whole Noxa protein when cross-linked with pHSP27 and the peptide comprising the N-terminal region without the cross-linking Cys25 equally delayed the proteasomal degradation of ubiquitylated IκBα. Therefore, the role of pHSP27 is likely to expose the N-terminal region of Noxa to form a complex with IκBα and NF-κB. HSP27 has vastly different functions, including reducing growth or proliferation and increasing differentiation, and protecting against apoptosis.^[@R36]^ This diverse function of HSP27 may depend on its ability as a chaperone protein to interact with other proteins that are present in various cell types or conditions. Further, HSP27 can be phosphorylated at three serine residues, resulting in the redistribution of the large oligomer into smaller tetrameric units, and its dephosphorylation favors the formation of large oligomers, the dimer of HSP27 being the building block for multimeric complexes that can be up to 1000 kDa.^[@R37]^ It is possible that depending on the phosphorylation state, HSP27 interacts with different proteins to ultimately mediate different outcomes. For example, in prostate cancer cells, HSP27-eIF4E interaction defines the chaperoning role of HSP27 to decrease eIF4E ubiquitylation and proteasomal degradation to protect the protein synthesis initiation process and survival.^[@R38]^ Similarly, hic-5 interacts with HSP27 and blocks the anti-apoptotic role of HSP27.^[@R39]^

Our finding that pHSP27 has an anti-inflammatory role is consistent with the report that HSP27 deficiency in mice augments neutrophil infiltration in wounds.^[@R40]^ In other studies, HSP27 can enhance NF-κB activity by interacting with IKK ^[@R41]^ or by interacting with polyubiquitin chains.^[@R42]^ However, these studies focused on analyzing the presence or absence of HSP27 and did not investigate the phosphorylation state of HSP27. Our studies demonstrate a novel observation that Noxa expression being highly induced in airway epithelial cells may define the anti-inflammatory function of pHSP27.

The finding that pHSP27 is primarily detected in lung tissues or airway epithelial cells would suggest that the anti-inflammatory role of Noxa is restricted to AECs. Consistent with this hypothesis, inducible Noxa expression in airway epithelial cells of transgenic mice was effective in suppressing inflammation. Delivering the NoxaA peptide directly to the airways may be a useful tool to temper inflammatory responses by prolonging the half-life of IκBα. Therefore, developing therapeutic vectors that deliver Noxa proteins or peptides to the airway epithelium or molecules that specifically induce Noxa expression in airway cells would represent novel anti-inflammatory tools to control chronic inflammation. Noxa as treatment may be effective particularly in diseases that are associated with high levels of pHSP27, including COPD,^[@R43]^ asthma,^[@R44]^ and cancer.^[@R45]^ IFN-γ has been used to treat chronic inflammatory diseases, but as a pleiotropic cytokine, it has many effects. By identifying Noxa as the protein that mediates the anti-inflammatory role of IFN-γ this study may have uncovered a means to harness the anti-inflammatory benefits of IFN-γ and minimize the other effects of IFN-γ This would place Noxa in a unique position among anti-inflammatory therapeutic agents, especially when prolonged treatments are expected in chronic inflammatory diseases, such as asthma and COPD.

Materials and Methods {#S11}
=====================

Animals {#S12}
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All mouse studies were performed at the Lovelace Respiratory Research Institute (LRRI), a facility approved by the Association for the Assessment and Accreditation for Laboratory Animal Care International using protocols, pre-approved by the Institutional Animal Care and Use Committee (IACUC), and the Environmental Safety and Health department (ES&H). *Noxa*^-/-^ on C57Bl/6 background mice were provided by Dr. A. Strasser (Walter and Eliza Hall Institute, Melbourne, Australia) and *p53*^-/+^ breeders were purchased from The Jackson Laboratory (Ben Harbor, ME) and were bred at LRRI.^[@R17]^ Mice were housed in isolated cages under specific pathogen-free conditions for the described studies.

The conditional and airway specific overexpression of Noxa was achieved by mating two lines of transgenic mice, the CCSP--reverse tetracyclineresponsive transactivator (rtTA) mice, bearing the rtTA under the control of the CCSP gene promoter (Perl et al 2009), and the tetracycline operator (TetO)~7~--Noxa mice, containing TetO and minimal cytomegalovirus (CMV) promoter and the Noxa transgene. (TetO)~7~-Noxa mice were generated at the M.D. Anderson Genetically Engineered Mouse Facility following standard methods and Institutional Animal Care and Use Committee-approved protocols and bred with CCSP-rtTA mice at LRRI. The transgene plasmid was cut with HindIII and NheI, and murine Noxa cDNA was separated from the TG-1 vector. Purified murine Noxa cDNA was injected into the pronuclei of zygotes collected from superovulated (C57BL/6) female mice, and injected zygotes were transferred to pseudopregnant ICR recipient females. Founder animals were identified by polymerase chain reaction amplification of 2.5 ul of tail DNA segments digested overnight in NaOH at 55°C. Oligonucleotides were as follows: a, 5′-AGGGATCCAT GTGTATTGTA TGTGGGCTC TAACCTGGC-3′ b, 5′-TGCAGCTAGG TGAGCGTCCA CAGGACCCTG AGTGGT-3′. To conditionally express murine Noxa in the lung, transgenic mice were fed with doxycycline-containing diet. Founder animals (C57Bl/6J) were bred to C57Bl/6J × C57Bl/6J animals to evaluate germline transmission. Six lines that transmitted the Noxa sequence were analyzed by q-PCR for expression of Noxa in lung and tracheal tissue. On the basis of these results, three murine lines with expression of Noxa in the airway epithelia were chosen for colony expansion and line 2 was maintained by breeding with wild-type C57Bl/6 mice and using the wild-type littermates as controls for each experiment.

Exposures {#S13}
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At 6-10 weeks of age, mice were entered into the experimental protocols. Mice were instilled with HDM extracts for 5 consecutive days and lung tissues were analyzed 24 or 72 h post the last challenge. HDM (Greer Laboratories, Lenoir, NC) was from *Dermatophagoides Pteronyssinus*, (lot \#248041), as 21.5 mg protein per 103.3 mg dry weight in a freeze-dried form, was resuspended in sterile phosphate buffered saline for a final concentration of 1μg/μl and stored in frozen aliquots at -20°C. Sensitization and exposure of mice to ovalbumin was as described previously.^[@R46]^ Bronchoalveolar lavage and preparation of lung tissues for histopathological examination and staining with Alcian blue/hematoxylin and eosin (H&E) was as described.^[@R46]^

Cells {#S14}
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Primary human airway epithelial cells (HAECs) (Cambrex Bio Science Walkersville, Inc.), murine airway epithelial cells (MAECs), AALEB cells, A549 cells stably transfected with multiple copies of the NF-κB response element driving a luciferase reporter construct (A549-NF-κB-luc cell line) (Panomics, Inc., CA.), and 293T cells were cultured as described previously.^[@R14]^ AALEB cells are immortalized HAECs that have been well characterized ^[@R47]^. Cells were maintained in bronchial epithelial growth medium BEGM (Lonza) supplemented with growth factors (BEGM Singlequots, Lonza) as described previously ^[@R14]^. Adenoviral expression vectors for Noxa were provided by Dr. G. Shore (McGill University, Montreal, Canada), and cells were infected as previously described ^[@R48]^. The A549-NF-κB-luc cell line was purchased from Panomics, Inc., CA. and grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 1mM sodium pyruvate, (Invitrogen), penicillin/streptomycin (Invitrogen) and hygromycin. The 293T cells were cultured in DMEM with 10% FBS. Transfections were carried out with the TransIT-2020 Transfection Reagent (Mirus Bio) according to manufacturer\'s protocol. Cell viability was determined by trypan blue exclusion.

Mouse airway epithelial cells (MAECs) were isolated essentially as described previously ^[@R49]^ by incubation in pronase solution (DMEM, 1.4 mg/ml pronase and 0.1 mg/ml DNase) overnight at 4°C to dissociate airway epithelial cells from the basal lamina. Cells were collected by gently rocking trachea in DMEM/Ham H12 media (Invitrogen) followed by centrifugation at 400g for 10 min at 4°C. MAECs were grown in media described previously.^[@R50]^

Generation of Expression Constructs {#S15}
-----------------------------------

The GST-tagged recombinant proteins of the full length Noxa (pGEX-Noxa), 5′ or 3′ truncated versions (pGEX-NoxaΔ5′ or pGEX-NoxaΔ3′), C25/51S mutant (pGEX-Noxa-2CS), and C-terminal K35/38/41R mutant (pGEX-Noxa-3KR) were expressed in pGEX-5X-1 Gene Fusion System (GE Health). The constructs were generated by amplifying the desired coding regions with appropriate mutations using PCR and inserting the products into the BamHI and EcoRI sites of pGEX-5X1 vector. The primers for generating NOXA full-length and truncated constructs were: NOXA (BamHI)- 5′-tcc gga ctc gga tcc cca tgc ct-3′ NOXA (EcoRI)- 5′-ccg tcg act gca gaa ttc tca gg-3′ NOXA-5′deletion (BamHI)-5′-cgg atc ccc caa ccg agc ccc gcg cgg gct cca gc-3′ NOXA-3′deletion (EcoRI) -- 5′-cag aat tct cac tgc cgg aag ttc agt ttg tct cc-3′. Primers for generating pGEX-Noxa-2CS were: pGEX-NOXA-BamHI-F (5′-aag gtc gtg gga tcc cca tgc ctg gg-3′), NOXA C25S-F (5′-ctg gaa gtc gag tct gct act caa ctc-3′), NOXA C25S-R (5′-gag ttg agt agc aga ctc gac ttc cag-3′), NOXA-C51S-EcoRI-R (5′-cga ccc ggg aat tct cag gtt cct gag gag aag agt ttg g-3′). After generating products PCR1 and PCR2 using primers pGEX-NOXA-BamHI-F / NOXA C25S-R, and NOXA C25S-F / NOXA-C51S-EcoRI-R, these products were purified and used as the templates to amplify PCR3 product using pGEX-NOXA-BamHI-F and NOXA-C51S-EcoRI-R primers. PCR3 was digested with BamHI+EcoRI, and cloned into pGEX-5X1. pGEX-Noxa-3KR was constructed using the following primers: pGEX-NOXA-BamHI-F, NOXA-K35R-R (5′-ctg ccg gaa gtt cag gcg gtc tcc aaa tct-3′), NOXA-K41R-R (5′-gat atc aga ttc aga agg cgc tgc cgg aag ttc ag-3′), NOXA-K48R-EcoRI-R: (5′-cgg gaa ttc tca ggt tcc tga gca gaa gag gcg gga tat cag att cag-3′). To express Noxa and its mutant proteins in mammalian cells, Noxa-WT, Noxa-2C and Noxa-3KR were sub-cloned into the BamHI and EcoRI sites of pcDβA vector. pcDβA-Noxa-C25S and pcDβA-C51S constructs were generated using pGEX-NOXA-BamHI-F/NOXA-EcoRI-R (5′-cga ccc ggg aat tct cag gtt cct gag cag aag agt ttg g-3′) to amplify pGEX-Noxa-2C and pGEX-NOXA-BamHI-F / NOXA-C51S-EcoRI-R to amplify Noxa-WT. The PCR products were digested with BamHI+EcoRI and sub-cloned into the pcDβA vector, where expression of cloned sequences is driven by the CMV enhancer. All constructs were verified by sequencing.

Immunofluorescence {#S16}
------------------

Cells were seeded onto 2- or 4-well chamber slides (Labtek), fixed with 3.5% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated in blocking buffer (3% BSA, 1% normal goat serum in PBS). After incubation with the relevant primary antibody cells were stained with either anti-rabbit Alexa647 or mouse Alexa555 (Invitrogen), mounted with glass coverslips using Fluoromount-G (Southern Biotech) and immunofluorescence visualized using Axioplan 2 (Carl Zeiss) with a Plan-Aprochromal 63×/1.4 oil objective and a charge-coupled device camera (SensiCam; PCO) using an acquisition and processing software Slidebook 5.0 (Intelligent Imaging Innovation, CO).

GST Pull-down Assays {#S17}
--------------------

Expression constructs were transformed into ECOS-21 bacterial cells (Yeastern Biotech) and induced using 1mM IPTG (Sigma) for 4 h at 37°C (GE Health, manufacturer\'s protocol). Cells were lysed by repeated freeze-thawing and sonication. Expression was determined via Western blot analysis using an anti-Noxa Mouse mAb (114C307, Calbiochem). Fusion proteins were affinity-purified on glutathione-Sepharose beads (Pharmacia) and quantified. For pull-down assays, lysates of non-treated and IFN-γ-treated cells were incubated for 2 h with GST fusion proteins noncovalently immobilized on glutathione-Sepharose beads. Beads were washed in cell lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 1 mM PMSF, 0.07 trypsin inhibitor units/ml of aprotinin, and 1 μg/ml each of leupeptin, pepstatin, antipain, and chymostatin (Sigma)). Bound proteins were solubilized in sample buffer, resolved by SDS-PAGE, and subjected to anti-HSP27 immunoblotting.

Preparation of Cytoplasmic and Nuclear Extracts {#S18}
-----------------------------------------------

Briefly, cells were lysed in 100 μl lysis buffer (10 mM Tris-HCl, pH8.0; 60 mM KCl; 1 mM EDTA; 1 mM DTT, protease Inhibitors cocktail, 0.5% NP-40), and nuclei centrifuged at 2000g. Nuclear extract was obtained by incubating nuclei in nuclear extraction buffer (20 mM Tris-HCl; 420 mM NaCl; 0.2 mM EDTA; 25% glycerol; 1.5 mM MgCl~2~; 1 mM DTT and 0.1 mM PMSF) and centrifugation at 10,000g.^[@R15]^

Immunoblotting {#S19}
--------------

Western blot analysis was performed as described previously ^[@R15]^. Briefly, equal amount of proteins were resolved by SDS-PAGE and transferred to PVDF membranes (Immobilon-P, Millipore Corp). Membranes were incubated with optimal concentrations of relevant primary antibody and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse or -rabbit secondary antibody (Sigma), washed 3×5 min in TBS-T and visualized by Western Lightning Plus-ECL according to manufacturer\'s protocol (Perkin Elmer). The antibodies used were mouse Noxa (114C307) (Calbiochem), rabbit anti-Noxa (FL-54), anti-κBα (C-21) and mouse anti-NF-κB p65 (F-6) (Santa Cruz Biotechnology), rabbit anti-Lamin A/C and mouse HSP27 (Cell Signaling Technology), rabbit anti-p-Ser ^15,\ 78,\ or\ 82^ HSP27 (Stressgen), anti-K48-linkage Specific Polyubiquitin mAB (Cell Signaling), mouse anti-β-Actin, and anti-β-tubulin (Sigma).

Immunoprecipitation {#S20}
-------------------

The Crosslink IP Kit (Thermo Fisher Scientific) was used to cross-link 10 μg of anti-Noxa, anti-NF-κB and anti-κBα (Santa Cruz) or HSP27 (Stressgen) antibodies to Sepharose protein A beads using disuccinmidyl suberate according to manufacturer\'s protocol. The associated proteins were immunoprecipitated by gentle mixing equal amounts of protein lysates with the antibody-crosslinked beads at 4°C overnight. After 6 washes, bead-bound proteins were eluted and analyzed by immunoblotting.

Retroviral silencing with shRNA {#S21}
-------------------------------

Retroviral silencing vector encoding shHSP27 or shNoxa and the corresponding control vector (shCtrl) were purchased from Origene Technologies, Inc. The suppressing effect of the shRNA was first established in AALEB cells and the subsequent amplification, purification, and packaging of the retroviral particles using Phoenix cells (Orbigen, Inc.) were performed as specified by the manufacturer.

Luciferase assay {#S22}
----------------

A549 /NF-κB-luc cell line (Panomics Corp.) was used to monitor the activity of NF-κB transcription factor in a cell based assay (R/D Biosystems, Minneapolis, MN). Cells were lysed by rocking for 15 min in passive lysis buffer (Promega Corp., Madison, WI) and luciferase activity was detected with the Luciferase Assay system (Promega) using a Fluoroskan Ascent detector (Labsystems) according to the manufacturer\'s protocol.

Luminex Cytokine Assay {#S23}
----------------------

The cytokines in cell culture media were quantified by Luminex instrument (Luminex Corp.) using Multiplex Fluorescent Bead-Based Luminex Cytokine Assays (EMD Millipore).

MALDI-TOF {#S24}
---------

To identify interacting proteins, Noxa was immunoprecipitated with antibodies cross-linked to Sepharose protein A beads. The immunoprecipitate was fractionated on a SDS-PAGE and the 32 kDa band along with blank controls excised, eluted, and subjected to tryptic digestion. Samples were dissolved in methanol, mixed at a 1:1 ratio (v/v) with 2,5-dihydrobenzoic acid (20 mg/ml in 70% methanol in water) as matrix. Survey scans were acquired from *m*/*z* 500--4,000. Sequence analysis of the tryptic peptides was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (ABI 4700 MALDI /TOF Mass Spectrometer) running 4000 Series Explorer. Precursors for MS/MS were selected on the basis of signal intensity with up to 20 peptides per spot being chosen for MS/MS analysis. Peptide sequences were identified by searching MASCOT® database using GPS Explorer Version 3.5 software (Applied Biosystems). Both MS and MS/MS spectra were processed by the software. For MS spectra, an S/N threshold of 30 was used, while for MS/MS spectra, a threshold of 20 was used to detect peaks.

Real-time PCR Analysis {#S25}
----------------------

Total RNA was isolated from HAECs, MAECs and AALEB cells using RNeasy Micro Kit (QIAGEN). The mRNA levels of Noxa, IL-1β, IL-6, Bcl-2, Bcl-x~L~ were quantified by ABI HT 7900 Real Time PCR system using TaqMan One-step RT-PCR Gene Expression kit (Life Technologies). The mRNA levels of KC from MAECs were quantified using High Capacity RNA-to-cDNA Kit (Life Technologies) and QuantiTect SYBR Green PCR Kit (QIAGEN).

Statistical Analysis {#S26}
--------------------

Grouped results from at least three different experiments were expressed as means ± SEM. Results (grouped by time point and treatment) were analyzed using two-way analysis of variance. In the event that significant main effects were detected (*P* \< 0.05), Fisher\'s least significant difference test was used to differentiate between groups. A *P*-value of 0.05 was considered to indicate statistical significance. Data were analyzed using statistical analysis software (Statistical Analysis Software Institute).

Supplementary Material {#S27}
======================

######

**Figure S1. (A)** Primary normal human airway epithelial cells (HAECs) were treated with 0, 25, or 50 ng/ml IFN-γ for 24 and 48 h and analyzed for Noxa mRNA levels by qRT-PCR. **(B)** Induction of Noxa mRNA in MAECs from wild-type, *noxa^-/-^* and *STAT1^-/-^* mice treated with 50 ng/ml IFN-γ for 24 h. **(C)** IFNγ induces Noxa independent of p53. MAECs from *p53^-/-^* mice were treated with 50 ng/ml of IFN-γ for 48 h or left untreated and mRNA analyzed by qRT-PCR. **(D)** Susceptibility of *noxa^+/+^* and *noxa^-/-^* MAECs to 50 ng/ml IFN-γ treatment over 96 and 120 h. Error bar indicates ± SEM (n=3 wells/group) \* *P* \< 0.05.

**Figure S2. (A)** Mucous cells/mm basal lamina were higher after exposure to ovalbumin in *noxa^-/-^*compared with *noxa^+/+^* mice. **(B)** Perivascular inflammation in *noxa^+/+^* and *noxa^-/-^* mice exposed to allergen for 5 as scored by a person unaware of slide identities. Mice were injected IP with OVA/Alum on days 1 and 7 and exposed to OVA aerosols at 2 mg/m^[@R3]^ for 5 d (n=6 mice/group). **(C)** AALEB cells were treated with increasing concentrations of H~2~O~2~ and cell counts were determined. Error bars indicate ± SEM, \* *P* \< 0.05. **(D)** AALEB cells infected with Ad-Noxa or Ad-GFP at either 0, 20, 30, or 40 MOI and treated 24 h later with 45 μM H~2~O~2~ (n= 3 wells per group) **(E)** A549-NF-κB-luc were infected with either Ad-GFP or Ad-Noxa at 100 MOI for 16 h and or treated with 50 ng/ml human recombinant IFN-γ or vehicle for 15 h. Subsequently cells were treated with 10 ng/ml TNFα for 6 h and analyzed for luciferase activity. Luciferase values were corrected for total protein. (n= 3 wells per group) **(F)** AALEB cells treated with vehicle or IFN-γ were treated with TNFα for 30 min and following Western blotting were probed for NF-κB, IκB, Noxa, β-tubulin, and lamin A/C. **(G)** Cytosolic extracts prepared from *noxa^+/+^* and *noxa^-/-^*MAECs treated with TNFα for 0, 15 and 30 min were analyzed by Western blotting. The extracts were probed for NF-κB (p-65), IκB, and β-actin, and the relative fold-change of cytosolic IκBα was quantified over the respective β-actin levels. Error bar indicates ± SEM (n=3 independent experiments with 3 wells in each experiment) \*denotes *P* \< 0.05, \*\*\* denotes *P* \< 0.001.

**Figure S3: (A)** AALEB cells infected with Ad-Noxa or Ad-GFP (40 MOI) for 24 h and then treated with IFN-γ or nothing as control were subjected to immunoblot analyses using anti-Noxa and anti- p-S~82~HSP27 antibodies. **(B)** GST-Noxa-coated beads incubated with extracts from IFN-γ-treated AALEB cells in the presence or absence of 5 mM DTT for 6h. The pull-down products were analyzed by Western blotting using antibodies to Noxa and GST. **(C)** GST or GST-Noxa proteins bound to glutathione-sepharose beads were used to pull down interacting proteins from AALEB cell lysates and analyzed by Western blotting using antibodies to p-S~82~HSP27, Noxa, and β-actin. **(D)** shHSP27 and shCtrl cells treated with IFN-γ and tested for Noxa expression by Western blotting using antibodies to Noxa and β-actin. **(E+F)** Multiplex bead-based Luminex assays of cytokines in shControl, shNoxa and shHSP27 AALEB cells. AALEB cells stably transfected with shCtrl, shNoxa, or shHSP27 were pretreated with IFN-γ for 24 h and either treated with TNFα (20 ng/ml) or were left untreated for 2 h. Culture media supernatants from at least 3 wells per group were subjected to Multiplex Bead-based Luminex assays for IL-8 **(E)** and IL-6 **(F)**. Error bar indicates ± SEM \*\*\* *P* \< 0.001.

**Figure S4. Co-localization of Noxa, NF-κB and HSP27 *in vivo*. (A)** Immunostaining with Noxa (red) and pS~82~Hsp27 (green) antibodies of the airway epithelium from *Noxa^WT^*and *Noxa^Ind^* mice following HDM challenge and dox-mediated Noxa induction. **(B)** Immunostaining with NF-κB (green) and pS~82~Hsp27 (red) antibodies of the airway epithelium from *Noxa^WT^*and *Noxa^Ind^* mice.

**Figure S5: (A)** The amino acid sequence of Noxa is highly conserved for mouse and human. Murine Noxa appears to have a duplicate of the human sequence. Especially, the N-terminal sequence and the BH3 domain are conserved and represented twice in the murine Noxa. **(B)** Amino acid sequences of NoxaA, NoxaB, NoxaC, and NoxaD peptides. **(C)** HAECs were treated with 45 μM H~2~O~2~ and 1.0 μM Noxa C peptide and cell counts were determined after 24 h. Error bars indicate ± SEM.

**Table S1.** Peptide mass fingerprint analysis by Matrix Assisted Laser Desorption Ionization-Time of Flight mass spectrometry (MALDI-TOF). Protein extracts from AALEB cells treated with IFN-γ for 48 h were immunoprecipitated using Noxa antibodies, fractionated by PAGE. After elution from the gel, the 32 kDa band was subjected to protease treatment and MALDI-TOF analysis. These are data related to [Figure 2](#F2){ref-type="fig"}.

The authors thank Dr. Ram H. Nagaraj for providing HSP27 WT and C137A expression constructs and Lois K. Herrera for technical assistance with Luminex assays and quantification of inflammation in lung tissues. These studies were supported by grants from the National Institutes of Health (HL068111 and ES015482).

**Author Contributions:** CZ performed most of the experiments and drafted parts of the paper, JJ conducted the peptide, HDM, and ubiquitylation studies, HC performed the immunofluorescence studies, MW provided strategies to generate various mutation constructs, JX propagated the adenoviral constructs, YM helped with the mouse studies, YT designed the studies and wrote the manuscript for review by all co-authors.

**Disclosure/Conflict of Interest:** The authors have no conflicting financial interests.

![IFN-γ Induces Noxa Expression to inhibit inflammation\
**(A)** AALEB cells treated with IFN-γ or vehicle over 48 h and analyzed for Noxa mRNA by q-PCR. Data represented as the mean ± SEM (n= 3 wells/for each time point and repeated at least 3 times). **(B)** Protein extracts from HAECs treated with IFN-γ or vehicle and Noxa protein levels analyzed by Western blotting (representative of 3 experiments). **(C)** HAECs from 14 individuals treated with 50 ng/ml IFN-γ and analyzed for Noxa mRNA levels by q-PCR. **(D+E)** The *noxa*^+/+^ and *noxa^-/-^* mice challenged with HDM extracts (50 μg) intranasally in 50 μl saline for 4 days (n=5 mice/group) and the lung inflammatory cells quantified in the bronchoalveolar lavage (BAL) fluid collected 24 h later. BAL cell differentials quantified after staining with Wright Giemsa. The number of total cells **(D)** and eosinophils, macrophages, neutrophils, and lymphocytes **(E)**. Error bars indicate ± SEM, \* *P \<* 0.05, \*\* *P \<* 0.01, \*\*\* *P \<* 0.001.](nihms925046f1){#F1}

![Noxa blocks TNFα-induced NF-κB nuclear translocation and promoter activation by prolonging the half-life of IκBα: (A)\
AALEB cells were infected with Ad-Noxa or Ad-GFP (80 MOI) and were treated or left untreated with 20 ng/ml of TNFα for 24 h. Secreted IL-8 was quantified by Luminex assay (n= 3 experiments with 3 wells in each experiment). **(B)** Luciferase activity in A549-Luc cells when either infected with 100, 200 or 300 MOI Ad-Noxa or 300 MOI Ad-LacZ and stimulated with TNFα for 30 min, or **(C)** when the cells were stably transfected with shNoxa or shCtrl and treated with IFN-γ; reduced expression of Noxa mRNA and protein by shNoxa was verified by qRT-PCR and Western blotting. **(D)** AALEB cells treated with IFN-y (50 ng/ml) or vehicle and 24 h later with TNFα for 30 min. Cells immunostained with antibodies to NF-κB and Noxa and counterstained with Hoechst 33342. **(E)** Immunostaining with antibodies to NF-κB and Noxa of AALEB cells infected with Ad-Noxa or Ad-LacZ treated with TNFα for 30 min; percentage of cells with nuclear NF-κB. Error bar indicates ± SEM (n=3 independent experiments with 3 wells in each experiment). \* *P \<* 0.05, \*\* *P \<* 0.01, \*\*\* *P*\<0.001. **(F)** Cytosolic and nuclear extracts from *noxa^+/+^* and *noxa^-/-^* MAECs treated with IFN-γ and TNFα and analyzed by Western blotting. **(G)** AALEB cells infected with Ad-Noxa and Ad-GFP and treated with cyclohexamide. Extracts probed for NF-κB, IκB, Noxa, and GAPDH. **(H+I)** AALEB cells infected with Ad-GFP or Ad-Noxa, treated with 10 μM MG-132 and 30 min later with vehicle or 10 ng/ml TNFα; cells harvested 10 min later and extracts immunoprecipitated with anti-IκBα antibodies. **(H)** The levels of p-IκBα and Noxa levels were determined with the respective controls in input and immunoprecipitates. **(I)** Levels of p-IκBα and Noxa levels in the input and IκBα and K48 chain-linked ubiquitin of IκBα were determined in the immunoprecipitates. Western blots are representative of 3 different experiments.](nihms925046f2){#F2}

![The cross-linking of Noxa and p-HSP27 is essential for blocking NF-κB activation\
**(A)** Noxa (6 kDa) and a 32 kDa cross-reactive protein detected by Noxa antibodies in IFN-γ-treated AALEB cells. **(B)** Immunoprecipitates using anti-Noxa antibody from IFN-γ-treated cells analyzed by immunoblotting. **(C)** Immunoprecipitates using IgG as control or p-S~82~HSP27 antibodies from IFN-γ-treated AALEB cells analyzed by immunoblotting with antibodies to p-S~82~HSP27 and Noxa. **(D)** GST or GST-Noxa proteins bound to glutathione-sepharose beads used to pull down proteins from AALEB cell lysates and analyzed by Western blotting. **(E)** Protein extracts from AALEB cells transfected with HSP27 expression vector and treated with the p38MAPK inhibitor, SB203580, were immunoprecipitated using anti-Noxa antibodies. **(F)** HSP27 was co-transfected with Noxa, NoxaC25S, NoxaC51S, Noxa2CS, or Noxa3KR and immunoprecipitated using anti-p-S~82~HSP27. **(G)** Immunoprecipitates using Noxa antibodies from lysates of HEK293T cells expressing HSP27^WT^, HSP27C^137A^, and Noxa analyzed by Western blotting. **(H)** Immunofluorescence of shCtrl and shHSP27 cells treated with IFN-γ and/or TNFα or vehicle using antibodies to NF-κB and p-S~82~HSP27 and counterstained with Hoechst 33342. **(I)** Immunoprecipitates using anti-NF-κB from shCtrl and shHSP27 cells treated with IFN-γ and TNFα or vehicle and analyzed by immunoblotting. **(J)** Lysates from shCtrl and shHSP27 cells pre-treated with CHX and 10 ng/ml TNFα analyzed by Western blotting. All Western blots are representative of at least 3 different experiments.](nihms925046f3){#F3}

![Inducible transgenic expression of Noxa in airway epithelial cells protects mice from inflammation: (A)\
pHSP27 levels in HAECs, MAECs, MEFs and in murine lung, thymus, and liver tissues tested for by Western blotting. **(B)** Immunostaining with Noxa antibodies of tissue sections from the left lungs of Noxa^Ind^ and Noxa^WT^ littermates. **(C)** Noxa protein and mRNA levels in the right lungs analyzed by Western blottin and q-PCR, respectively. **(D)** Noxa^Ind^ and Noxa^WT^ mice were challenged with HDM extracts intranasally for 5 days, fed dox diet for 2 days, and lungs lavaged 24 h later (n=8 mice/group). The number of total cells, eosinophils and lymphocytes were quantified. **(E)** Mucous cell numbers per mm basal lamina (BL) were quantified using VisioMorph system on Alcian blue/hematoxylin and eosin (AB/H&E) stained sections (n=8/group). Representative images of AB/H&E-stained airway epithelium from Noxa^Ind^ and Noxa^WT^ mice. **(F)** Co-immunostaining with Noxa (red) and NF-κB (green) antibodies of airway epithelium from Noxa^Ind^ and Noxa^WT^ littermates following HDM challenge and dox-diet. Quantification of percentage airway epithelial cells with nuclear NF-κB in Noxa^Ind^ and Noxa^WT^ mice exposed to HDM (n=5/group). Error bars indicate ± SEM \* *P* \< 0.05,](nihms925046f4){#F4}

![N-terminal peptide of Noxa is sufficient to prolong IκBα half-life and block inflammation\
**(A)** Luciferase activity in A549-Luc cells treated with vehicle or peptides A, B, C, and D at 50 nM concentration. **(B)** Nuclear localization of NF-κB (p65) following treatment of *noxa^-/-^* MAECs with 0 or 10 ng/ml TNFα in the presence of TAT-Ctrl, and peptides A or D. **(C + D)** C57Bl/6 mice exposed to 50 μg house dust mite for 5 consecutive days and instilled with 10 μM each of TAT-Ctrl or peptide A on days 6 and 7. On day 8, the numbers of total inflammatory cells (C) and eosinophils **(D)** were assessed in the BAL fluid. Error bar indicates ± SEM (in 2 separate experiments with minimum of 4 mice/group each). \* *P* \< 0.05. **(E)** AALEB cells were treated with TAT-Ctrl or peptide A and 24 h later treated with TNFα for 10 min. Immunoprecipitates with anti-IκBα antibodies were analyzed for levels of IκBα and K48 chain linked ubiquitin of IκBα and IκBα and β-actin in the input. Western blots are representative of 3 different experiments.](nihms925046f5){#F5}

[^1]: Present address: Department of Oncology, Johns Hopkins Bayview Medical Center, Baltimore, MD 21224, USA

[^2]: Present address: Department of Immunology, Herbert Wertheim College of Medicine, Florida International University Miami, FL 33199, USA

[^3]: These authors contributed equally.


2012 European Athletics Championships – Men's 800 metres

The men's 800 metres at the 2012 European Athletics Championships was held at the Helsinki Olympic Stadium on 27, 28 and 29 June.

Medalists

Records

Schedule

Results

Round 1
First 4 in each heat (Q) and 4 best performers (q) advance to the Semifinals.

Semifinals
First 2 in each heat (Q) and 2 best performers (q) advance to the Final.

Final

References

Round 1 Results
Semifinal Results
Final Results

800
Category:800 metres at the European Athletics Championships


SP&M Article Archive

School Planning & Management's vast database of articles is a valuable tool for anyone who plans, designs, equips, maintains and operates pK-12 schools nationwide. Popular topics include facilities, safety & security, technology, business, finance and the learning environment. Articles are displayed in chronological order, beginning with the most recent.

Even the most thoughtfully designed and perfectly installed access control strategies can often easily be compromised by staff who do not do their part to support them. While staff development is called for, making access control a reality in deed as well as word is must be a leadership priority.

In recent years, more research has come to light in support of the impact that facility design has on student performance. These studies reveal clear evidence that the physical characteristics of a classroom impact learning, and furthermore, that a link exists between well-designed school facilities and increased academic achievement.

Charter schools are not for everyone and are meticulously designed for their student populations. As conservative school choice activists argue for greater funding for charter schools, like public schools, they differ in performance and student outcomes based on zip codes.

Technology has become an integral part of K-12 classrooms. Here are some examples of how districts across the country use technology—both software and hardware—to create authentic learning experiences for students.


In 1977, the Du Toit family purchased Die Pastorie (The Parsonage)-one of the most recognised properties in Riebeek Kasteel, from the Dutch Reformed Church. Thirty-three years later, Pieter and Annalene du Toit transformed this landmark property into one of the Riebeek Valley’s most luxurious overnight accommodation options, the Kloovenburg Pastorie.
This historic property has been affectionately restored and transformed into a beautiful villa, offering guests to the Riebeek Valley three sumptuous air-conditioned en-suite bedrooms and one super luxurious suite with en-suite bathroom and private lounge area. Along with the luxurious accommodation, the Kloovenburg Pastorie also has relaxing public spaces, including a well-appointed lounge, dining room, kitchen and a sparkling pool with loungers to relax on. The décor in this classic Victorian-styled home is an eclectic mix of French and Victorian.


Is hemifacial spasm a phenomenon of the central nervous system? --The role of desflurane on the lateral spread response.
A signature EMG feature of hemifacial spasm (HFS) is the lateral spread response (LSR). Desflurane is a common anesthetic with potent effects on synaptic transmission. We tested the hypothesis that the LSR is mediated by corticobulbar components by comparing the LSR during total intravenous anesthesia (TIVA) or TIVA plus desflurane during microvascular decompression (MVD) surgery. 22 HFS patients undergoing MVD surgery participated in this prospective study. The LSR data was recorded from the o. oculi, o. oris and mentalis muscles prior to opening dura. LSR onset latencies and amplitudes were determined under TIVA and TIVA/desflurane (0.5 and 1MAC). Facial muscle LSRs and EEG were analyzed. Desflurane (1MAC) significantly decreased the LSR amplitude in all 3 facial muscles (p<0.01). Pooled LSR data from all facial muscles showed desflurane inhibited the LSR amplitude by 43% compared to TIVA (p<0.001). No effects on the latency of the LSR or on EEG state were observed. LSR inhibition by desflurane suggests a central mechanism involvement in the genesis of this signature HFS response. This study demonstrates that facial nerve vascular compression and plastic changes within the CNS are part of the pathophysiology of HFS.


651 F.2d 176
18 ERC 2128, 11 Envtl. L. Rep. 20,857
ROHM & HAAS COMPANY, Appellant,v.UNITED STATES ENVIRONMENTAL PROTECTION AGENCY, and Walter C.Barber, and Mobil Oil Corporation, Appellees.
No. 81-1757.
United States Court of Appeals,Third Circuit.
Argued May 22, 1981.Decided May 26, 1981.

James J. Binns, Philadelphia, Pa., Patrick M. Raher, David J. Hayes, David A. Kikel, Washington, D.C., for appellant Rohm and Haas Company; Robert P. Vogel, Regulatory Counsel, Rohm and Haas Company, Philadelphia, Pa., of counsel.
David Richman, Pepper, Hamilton & Scheetz, Philadelphia, Pa., A. Duncan Whitaker, Mark D. Wegener, Ray A. Jacobsen, Jr., Howrey & Simon, Harold Himmelman, Cynthia A. Lewis, Beveridge & Diamond, Washington, D.C., for intervenor-defendant Mobil Oil Corporation; Thomas J. Winans, Deborah E. Lynch, Mobil Oil Corp., New York City, of counsel.
Before GIBBONS, GARTH and SLOVITER, Circuit Judges.
OPINION OF THE COURT
PER CURIAM.

1
Rohm & Haas Company appeals from a judgment in favor of the defendants in its action against the Environmental Protection Agency et al., in which Mobil Oil Corporation intervened as a defendant.  Rohm & Haas sought an injunction against the issuance to Mobil of an Experimental Use Permit (EUP) for experimental use of Mobil's product Tackle as a pesticide for soybeans.  Rohm & Haas contends, and the defendants do not dispute, that the Environmental Protection Agency, in approving experimental use of Tackle, relied on information in the agency's file with respect to Rohm & Haas's product Blazer, which like Tackle contains the active ingredient acifluorfen.  Such reliance, Rohm & Haas contends:

2
(1) violates Section 5 of the Federal Fungicide and Rodenticide Act, 7 U.S.C. § 136c in that EUP's may not be issued in reliance on data submitted by anyone but the applicant; and

3
(2) violates restrictions on the use of submitted data in Section 3(c)(1)(D) of the Federal Fungicide and Rodenticide Act, 7 U.S.C. 136a(c)(1)(D).

4
Rohm & Haas also contends that the agency should have permitted additional time for public comments in response to its May 5, 1981 Federal Register Notice of Mobil's application for the EUP.

5
Upon filing its notice of appeal Rohm & Haas moved for an injunction pending appeal, and alternatively for accelerated disposition of the appeal after oral argument, on the briefs filed with the trial court.  All parties agreed to a disposition of the appeal on the submissions to date.  We heard oral argument on the merits of the appeal on May 22, 1981.

6
We affirm the judgment appealed from, essentially for the reasons set forth in the comprehensive opinion of the trial court.


Q:

Inno setup: how to replace a string in XML file?

The following (in quote) is the content of a XML file which is part of my package.
I'd like to replace the value of c:\path\myapp.exe during the installation (with the real path where the user chose to install the application.
Is that possible? How to?
<?xml version="1.0" encoding="UTF-8"?>
<launchConfiguration type="org.eclipse.ui.externaltools.ProgramLaunchConfigurationType">
   <listAttribute key="org.eclipse.debug.ui.favoriteGroups">
      <listEntry value="org.eclipse.ui.externaltools.launchGroup"/>
   </listAttribute>
   <stringAttribute key="org.eclipse.ui.externaltools.ATTR_LAUNCH_CONFIGURATION_BUILD_SCOPE" value="${none}"/>
   <stringAttribute key="org.eclipse.ui.externaltools.ATTR_LOCATION" value="c:\path\myapp.exe"/>
   <stringAttribute key="org.eclipse.ui.externaltools.ATTR_TOOL_ARGUMENTS" value="${resource_loc}"/>
</launchConfiguration>

A:

Your best option is to use the XML DOM to select and edit the required node as TLama suggested.
Alternatively, you can install a template file with a known string in the location you want to replace.
The file can then be read as a string, modified and written back out again using something like:
[Code]
procedure WriteAppPath;
var
    FileData: String;
begin
    LoadStringFromFile(ExpandConstant('{app}\app.xml'), FileData);
    StringChange(FileData, 'XXXXXMARKERXXXXX', ExpandConstant('{app}'));
    SaveStringToFile(ExpandConstant('{app}\app.xml'), FileData, False);
end;

See also this question about doing the same thing en masse to an INI file.


Ultrasensitive electrochemical immunosensor for clinical immunoassay using thionine-doped magnetic gold nanospheres as labels and horseradish peroxidase as enhancer.
A new signal amplification strategy based on thionine (TH)-doped magnetic gold nanospheres as labels and horseradish peroxidase (HRP) as enhancer holds promise to improve the sensitivity and detection limit of the immunoassay for carcinoembryonic antigen (CEA), as a model protein. This immunoassay system was fabricated on a carbon fiber microelectrode (CFME) covered with a well-ordered anti-CEA/protein A/nanogold architecture. The reverse micelle method was initially used for the preparation of TH-doped magnetic gold nanospheres (nanospheres), and the synthesized nanospheres were then labeled on HRP-bound anti-CEA as a secondary antibody (bionanospheres). Sandwich-type protocol was successfully introduced to develop a new high-efficiency electrochemical immunoassay with the labeled bionanospheres toward the reduction of H2O2. Under optimized conditions, the linear range of the proposed immunoassay without HRP as enhancer was 1.2-125 ng/mL CEA, whereas the assay sensitivity by using HRP as enhancer could be further increased to 0.01 ng/mL with the linear range from 0.01 to 160 ng/mL CEA. The developed immunoassay method showed good precision, high sensitivity, acceptable stability and reproducibility, and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay (ELISA) method.


"I didn't want to..." "I'm not in control." "I know." "Tell me you love me." "I love you." "Ross, darling." "What?" "You're afraid I'll go deaf?" "Get up, I'm driving you to school." "You got an other one?" "You're a minor, it's not legal for you to get a tatoo and now you have two!" "Three?" "Where is the third one?" " Why are you so pissed at everyone?" " Not everyone." "Ok, me." "Why are you so pissed at me?" "You turned up at my door step." "I take you in, feed you, cloth you, send you to school." "But I suck, uh?" " I mean, I must really..." " You left me." "After mom..." "The victim of a Baghdad car bombing." "We did transplants, and facial reconstructions." "This triathlete was told he'd never compete again." "For some women, bigger is better." "What these people have in common is that they have each in some level, and for variant reasons, altered themselves." "But where is the threshold?" "When is it ok to intervene in God's work?" "This question is the very corner stone of bioethics and we're gonna spend the rest of the semester dealing with it." "Come on, Will, I mean it." "I'm a bartender and a dropout." " You're a professor." " And a surgeon." "How can you take me seriously?" " What makes you think I take you seriously?" " Do you?" " You're not kidding?" " Why are you with me?" " You're different." " In what?" "When your whole life is planed out for you while you're still in the womb, it kinda kills the mystery." "And then you came along and you were..." " unexpected." " Unexpected?" "The one choice my father didn't make for me." "I gotta to run." " Hum, we're still on for dinner?" " 8 o'clock?" "Make it 8:30, I have to go to the lab for a few hours." "While we are on the subject, why are you with me?" "You're not entirely uncharming." "It's not insulting." " I'm 16." " You're 15!" "And quit being melodramatic." " She's weird." " She's not that weird!" "I don't need a baby-sitter." "Don't think of me as a baby-sitter, think of me as prison guard." "I brought Parcheese." "I think there's a pretty good chance the grant could come true." "The institute indicates it's a done deal." "And I heard Paris is great in spring and..." " You're not happy?" " Of course I'm happy." "Because I think you should come with me." " I can't." " Why not?" " I have a job." " Jaime!" " A life." " We're talking Paris." "And I have reponsabilities." " Did I mention it's Paris?" " I'm pregnant." "Yeah, that's what I said." "I don't want you to..." "I just..." "I know this is not something you just spring on someone..." "Except for the part where that's exactly what I'm doing to you right now and..." "I know you're in a place where you can't even think about it..." "And god, I'm only 24!" "It's not, I mean..." "I said to myself that's the only one thing to do but..." " I will never be..." " 5 months 14 days..." "That's how long we've been dating." "Jaime, if somebody had asked me 5 months and 15 days ago, if I believed in love at first sight," "I would have said I absolutely did not." "And the very next day, I would have been proved them all." " Will..." " Marry me." "So I'm thinking Coletrane if it's a boy." "Billy if it's a girl." "You don't have to do this you know." "Actually, I'm pretty sure you have to name them." " I mean you don't have to..." " I know, and I'm still asking..." "So what does that tell you?" "Jaime?" "Jaime?" "There's internal bleeding." "Extensive crush injuries in right arm and both legs." " She's coming up!" " I need 5 milligrams of **." " Will?" " You're gonna be ok, baby." "We're gonna put you back to sleep." "It's gonna be alright." "I promise." "It's gonna be ok..." "Doctor Anthros, surely a more conventional hospital..." " Did you assemble the team?" " They're in the O.R. but I don't understand why..." "You don't have to!" "Multiple compound radial and humeral fractures on the right arm." "Severed brachial artery." " Right arm is a shredder." "We're looking at complete amputation on both legs." "There are vascular damages, it's catastrophic." " Lost the fetal heartbeat." " Doctor Anthros?" "BP is falling. 80 systolic." " Prep her." " Doctor Anthros?" "Do it!" "Why didn't somebody stop him?" "Sorry John, we can't." "He has full clearance and we didn't have the authority to stop him." "It's the price you pay for working with a so-called genius." "'Cause he must have left his I.Q. in the car." "Debriding the humeral nerve, radial nerve appears to be intact." "Should I intervene?" "No." "This operation and this patient doesn't officially exist." "We have deniability, it's a freebee." "We can always terminate later if need be." "Meanwhile get Jae." "See if you can track him down." "Forgive me Father for I have sinned." "The doctor." "Did you kill him?" "It was like a ride at Disneyland." " Which one?" " I've never been." "I'll take you." "You'll love it." "It's all... fat people with fat children walking along aimlessly." "Too stupid to know the difference." "Do you miss it?" "The way things were." "If I could remember, I'm sure I would." "Do I remind you of her?" "Yes, keenly." "Tell me you love me." " Jaime?" " What happened?" "You had an accident." "You're ok." "My baby?" "What about the baby?" "Oh God!" "There was no permanent damage." "You'll be able to get pregnant again." "Why can't I feel my legs?" "Ok, Jaime, I need to tell you something." "What's happening to me?" "I need to tell about what I did, and you need to listen to me." " I can't feel my arm." " Listen to me, Jaime." "Both your legs and your arm had to be replaced." "Also, your right ear and right eye." "Molecular machines, intrasites, have been substituted for one eight of your blood cells." "Now you should have lacerations bone deep, all over your face." "But the intrasites are healing you." "At an exponential rate." "All of you, we're making you better." "It was a dream." "The bionics enhance the width of human strength." "And then some." " What have you done to me?" " Jaime." " Why did you do this to me?" " You were going to die." "Sir." "Borned Jaime Wells Sommers, Van Horn, Iowa," "February 23rd 1983." "Mother deceased, cancer." "Father still a hawkeye." "She's was enrolled at Berkeley until last semester which is when her sister began to attend classes at the Dolton school for the deafs." "She's clean except for a sealed court record from 1998." "What's in it?" " It's sealed." " Unseal it." "I'm working on it." "I think she's a good candidate." "Well let's pretend I'm soliciting opinions, why?" "She's stable." "I've put up an old Minnesota multiphysic personality inventory." "I liked what I saw." "Plus the situation whith her sister shows loyalty." "And she's smart." "I found an old I.Q. test." " How did she do?" " A little better than you, sir." "Jae, can you train her?" "Well, the last one we screened, tested, made the psych evaluations and she was a soldier." "Look where that got us." "We've always talked about non-military applications." "Here is our chance." "Your opinion is invalid, you're too close to the subject." "There's from the guy who played house with the last one." "Let's get fresh data, I want you to sit her down." "Alright." "Yeah, I once had a wolf." "And a wolf only makes a good pet, if he thinks he's a dog." "And your girlfriend, we're keeping her here against her will." "How long until she realizes that no one can make or do anything against her will." "You're talking about killing a human." "I'm talking about killing a wolf." "You know what scares me about you?" "Your father." "He seemed normal too." "If I'm anything like my father, you just made a big mistake." "Alright, I have to get you to rehab before... rehabilitation." "I'm not the one performing human experiments and keeping people prisonner." "You rehabilitate yourself." "Booh." " I'm Ruth." " I'm pissed." "Good." "We're here to talk about our feelings." "How do you feel about me being captured against my will?" "I was speaking editorially." "Actually, we're here to talk about your feelings." "Something no one seems to have any regard for." "Curve balls will do that." "You can have all the contingency plans you like and then someone goes into something like this and then we all have to stay after school..." "And talk about our feelings." " Your feelings." " Right." "After all, feelings are what got us here in the first place." "You like to draw?" " True or false?" " I have a sister." "She's staying with your landlady." "She thinks you're skiing in Vail." "This is where you stayed." "It looks nice." "You had fun." "Except for the part where" "Will enjured his arm skiing into a tree." "Does this mean I'll be going home at some point?" "It means we have a contengency plan, should you be going home at some point." "We also have a plan, should you not?" "True or false?" "You can't teach an old dog new tricks." "You would have done the same thing." "You never met my wife, did you?" "For two years I watched her die in a hospital while they disassembled her." "And the whole time I knew all I had to do to save her was to bring her here, but saving my wife was not the mission." "Ruth said she's stable." "How long until she's combat ready?" "She's a civilian." "Everyone has to sing for their supper around here, Will." "Haven't you figured that out yet?" "How long until the rest of her implants come online?" " She likes to draw." " I don't care." "I think you'll care about this." "Who did they bury?" "I mean, did you ever see the body?" " Did you show this to anybody else?" " Just you." "Keep it that way." "I can't protect you in here." " But once you're on the outside..." " Why should I trust you?" "Because I'm the only one you can trust." "Go back to your life like none of this ever happened." " I'll work it out with them." " What about you?" "Go, now!" "Alright, so I'll have those faxed over to you by Friday afternoon." "And, we're still on for that meeting tomorrow?" "Okay, good." "Yeah?" "Mummy?" "There is a lady out there running very fast." "Like as fast as the car!" "Sweety, what did I tell you about making things up?" "I thought it was cool that a girl could do that." "That's all." " Let her go." " What the hell did you do?" "You want her, right?" "For the program, leave her alone." "I'll make her understand what's at stake," " just let me have time to..." " Time to what?" "Letting her go is not a bad idea." "If you want her loyalty, give her freedom." "Or the illusion of freedom." "Alright." "Everybody, stand down." " You're making a mistake." " Listen to me." "The accident wasn't an accident." "According to the autopsy, the driver's neck was broken an hour before the crash." "Which means someone else was driving that car." "We need to find out who." "Jaime, it's Will." "Jaime, if you're there, pick up the phone, I need to talk to you." "Jaime, please." "There's so many these things I want to say to you." "So many things," "I can't say on the phone." "There are things I need to tell you about." "Before they happen." "Nice of you to show up." "I'm sorry." "What for?" "For leaving and not telling you first." "How was skiing?" "It was fine." "Don't lie to me!" "Where were you?" "I can't tell you." "You failed me." "Coffee." "It's no smoking." "What are you?" "A forest ranger?" "Something like that." "It must be scary." "Alone in the forest, just you and the animals." "Not if they know who's in charge." "Hey!" "Wait up!" "***." "We both know you're working in the Supermax Prison." "A thousand feet below Florence." "We both know this is the place where they stick people when they outlive their usefulness." " I don't know who you are." " If you don't do exactly what I say," "I'm gonna carve your wife Barbara like ***." "Baby, it's me." "Do what he says." "How did you do it?" "Do you know what they call creatures that can live comfortably in environments considered lethal, say, for instance, a thousand feet below the Earth's surface?" " How did you do it?" " Extremophiles." "I'll do an impression of one for you, ready?" " How... did you do..." " What?" " How did I do what?" " That Sarah Corvus is still alive." "And you come all the way here to tell me that?" "Let me do another impression for you." "Impression of a man who's been stabbed in the back, stripped of his rights and thrown in a hole like a dog." "Sarah Corvus is a time bomb, we both know it." "Well, time bombs only matter to those who have time." "Good bye, Jae." "Please give my regards to Will." "Tell him Daddy says hello." "Guards?" "If I may make an observation, you seem way too innocent and sweet to be behind a bar." "And you should be out there with the rest of them." "Finally, I'm not the only person who feels that way." " What can I get you?" " Beer." " On the house." " Thank you." "Don't I know you?" "We met very briefly." "We didn't even actually meet." " Where?" " Guess." " Are you alright?" " Yeah." " Let me take a look at it." " No, it's ok." "Are you ok?" "Come on, your husband will never know." "***." "Are you alright?" "Let me help you." "Just breathe." "It's like that, the first time the ear and eye inputs come online." "It's too much information." "We have to learn how to focus it." "And turn it off on our will." "You're a fast healer." "The intrasites are doing their job." "Who are you?" "Who are you!" "Tell everyone Sarah Corvus says hello." "Yo?" "You're looking for something?" "You need something?" "Uh?" "Come here, baby." "I got what you need." "Please." "Please don't." "Don't." " Are you ok?" " Tell me what you put in my head?" " Jaime." " I almost killed a man." "I didn't even know what I was doing." "What did you put in my head?" "Microscopic chips have been implanted in your cerebral cortex." "My father developped a technology for the military." "The program was meant to help amputees in the Gulf." "Of course he saw the combat application immediately." "You made me into a soldier!" "You're hardwired for very highly specialized warfare, yes." "I was gonna kill him." "I would have..." "But you didn't, you're still in control." " And with a proper training..." " Training?" "Jaime, we're the only ones that can protect you." " Let me protect you." " How can you protect me if you're scared at me?" "I never meant for any of this to touch you, I didn't." "I just..." "I couldn't bare to lose you." "That isn't even me." "Yes, it is." " Hey." " Hey." "I have to get back to Becca." " I can't keep leaving her like this." " Jaime." "What do you people do anyway?" " It's complicated." " Try me." "The world is further along than everyone wants to admit." "Technology is at a point where science-fiction isn't fiction anymore." "We keep that technology from getting into the wrong hands." "Who gets to decide right from wrong?" "What about Sarah Corvus?" "Where does she figure in all this?" "Where did you hear that name?" "Where did you hear that name, Jaime?" "Will?" "Will?" " Sorry." " Just hold on, ok?" " Where is she?" " I don't know." "I need everyone." "Time out." "The intrasites in our blood can filter out any impurities in our lungs." " Fringe benefit of being a freak." " What do you want from me?" "Honestly, I'm not sure..." " Jogging partner?" " Who are you?" "Without being too melodramatic about the whole thing, I'm Sarah Corvus... the first bionic woman." "Tada!" "So what did they replace?" "I figured the eyes and the ears." "What else?" "Me, they did both arms, both legs, only one eye." "Anthros wasn't sure about the optical interface." "Neither was his son." "So I did the other one myself." "Part of my chest too." "I'm cutting away all the parts of me that are weak." "Time in." "You only have one bionic arm." "You should really do something about that." "You gotta have to do a little better than that." "How am I doing now?" "Not bad." "Not bad at all." "We have a 33 year old male, caucasian, a gunshot victim, wound to shoulder." "Request assistance from Mercy General." "Will gonna make it?" "Miss Sommers." " Just wanted to get a look at you." " Who are you?" "You have 50 million dollars worth of my property inside you, so I guess you could say I'm your landlord." "What do you want from me?" "I don't know, maybe you died 3 days ago and you just haven't realized that yet." "Is that a threat?" "There's no free agents, Miss Sommers." "Sooner or later you're gonna have to make a choice, it goes something like this, heads you lose, tails you die." "Welcome to the game." "Hey!" "If we do this, whatever this is, we do it on my terms." "If that's not okay with you," "I know what I'm capable of now." "So you send whoever you send, and I'll bury one guy after the next." "You understand me?" "By the way," "Sarah Corvus says hello." "Tell Jae we have a candidate." "What are you doing?" "I haven't seen the moon in a 1023 days." "It hasn't changed, let's go." "We have work to do." "To be continued..."


Author and political commentator on Fiji affairs, Rajendra Prasad, who authored Tears in Paradise – Suffering and Struggles of Indians in Fiji 1879-2004.

Corruption was the fastest growing industry during the
era of democratic rule in Fiji. It is not to claim that it has vanished but it
is being addressed. Much maligned Prime Minister Bainimarama and
Attorney-General Aiyaz Khaiyum have declared their assets, which gives
startling comparison to the wealth of leaders who were Prime Ministers of Fiji
except Rabuka who had the harvest but was not astute enough to hold it in his
granary. All others, except Rabuka, are multimillionaires. It clearly shows
that there is prestige, power and unlimited wealth that draw many, not through
genuine desire to serve, but to take advantage through such placement. Some say
Rabuka chose a different lifestyle and either misunderstood or underestimated
the treachery in Fiji politics. Once a hero to his people, following the
military coup of May 14, 1987 but today he is shunned and no political party
needs him or wants him. His plight is no different to George Speight except
that he is serving his sentence and Rabuka will remain a prisoner of his conscience
for the rest of his life.

In
the build up to the 2014 elections, racial attacks through the media or other
public forums, a common feature in the past elections, was demonstrably absent.
However, the Internet is being extensively used, sometimes savagely, where the
Blogs and Facebook sites give opportunity to people to rant and rave to their
hearts content. What is good about this
medium is that Internet is an even playing field where everyone can contribute
on issues of common interest. The voices of the weak cannot be stifled. Undoubtedly,
the political parties are also using this medium to their best advantage. But on the ground and in public, racial
comments and open display of racial hostility is not visible. Credit must go to
the Bainimarama Government for facilitating this remarkable change and for the
first time, equality and dignity of every citizen of Fiji is not only being
seen but felt across the nation. It has not only given dignity to Fijian
citizenship but also raised sense of patriotism in Fijians as never before.

FRANK BAINIMARAMA- credited with implementing policies to stem out racism and divisive politics, the fruits of which are evident in Fiji as witnessed by the Author.

It has contributed to a demonstrable change, as the two
dominant communities’ live in harmony and go about their day to day business
without rancour or bitterness. Go to the towns, markets or sports arenas, the
people of Fiji are one happy lot who relate well with one another. There is
happy and respectful exchange of greetings and hearty banter, followed by
laughter that is an evolving as Fiji Way, replacing the meaningless Pacific
Way. Letters to editor columns carry the same spirit and not the nasty
‘snarling’ of the past. Indo-Fijians
follow the Fiji Sevens team with same fervour as the iTaukei, including
Indo-Fijian women. Bollywood has also infiltrated the iTaukei hearts and minds,
drawing them to the TV screens and rigid following of their favourite
programmes. Go through the iTaukei settlements and it is not unusual to hear
the Bollywood beats echoing, as they do from Indo-Fijian homes. This medium is
also enhancing race relations, which is also transforming hearts and minds of
Fiji’s peoples, as they realize and accept they are one people and Fiji is
their home.

There
has also been a physical transformation, which cannot go unnoticed. ITaukei
girls, with their frail features and straightened hairs, adjusting to modern
fashion trends, are easily mistaken for Indo-Fijians. The effect of the roti
and curry across the nation is palpable.

Those muscular features
of the iTaukei are diminishing and the Policemen in sulu, without the bulging
calf muscles, look starved. On the Indo-Fijian front, the kava has contributed
to the diminishing physique. Indeed, there is physical harmony among the
peoples of Fiji that cannot be apprehended. However, Fiji can only become the
utopia of our dreams if there is political harmony. This can come about when
advocates of racism are disabled in the interests of the nation. Racism has
favoured a few who benefit from the spoils. Racism does not build but destroys
nations. Fiji has suffered too much from it for too long. Those closure of the
racial ‘kennels’ has brought about perceptible change that needs to be nurtured
with care, caution and thoughtfulness.

Indeed,
democracy in Fiji was a label without the basic ingredients of equality,
justice and dignity. Democracy that is reconfigured to pursue racial
discrimination is not democracy but tyranny. Both form and
content comprise its inseparable limbs. Worldwide such autocratic democracies
abound and it usually entails the tyranny of the aristocracy, propped by the Armed forces, against the
majority. Fiji has had a parting of ways between the two on December 5, 2006
and a new era in Fiji politics began, much to the disgust of the ruling elite
who had a long reign, using it to restore it to power whenever it was lost, as
in 1987 and 2000. With this detachment, a new era in Fiji politics began,
which will culminate in first democratic elections on September 17, 2014. The
field is open and those deposed in 2006 and their associates are back with
their ideologies and banners to retake what they considered to be their birth
right. Truth, morals, ethics and principles will become the immediate
casualties, as victory at any cost becomes the name of the game.

What
amused and also saddened me was the sheer lack of remorse and moral conscience
of some of the leaders, convicted for abuse of office or violation of taxation
laws, as they campaigned for their political parties. They moved around defiant
and dismissive of their past when common decency expected them to leave the
public domain. Indeed, the fodder of deceit and lies are
aplenty and the simple and gullible sometimes fail to distinguish between the
grain and chaff.

An unholy alliance, and marriage of convenience, where political enemies become friends for political expediency? [Fiji Sun Photo]

Sometimes, it is beyond the bizarre, as one person, a recent
arrival to the mainland from Vanua Levu told me that the Government was wrong
in fining Chaudhry F$2 million, as he was paying the farmers evicted from their
leased land $28,000 each from the funds he held in Australia! He genuinely
grieved that now the poor farmers cannot be helped because the Government had
taken all the money in fines. I was gobsmacked by the naivety of the person
whose seriousness in his belief was inscrutable. Will this election be won on
deceit and lies or will it be won on truth and understanding? Only time will
tell. God Bless Fiji!

[About the Author: Rajendra
Prasad is the author of Tears in
Paradise – Suffering and Struggles of Indians in Fiji 1879-2004), former Ba
Town Clerk and an analyst on Fiji’s struggling efforts to seek an appropriate
form of democracy.]


Issue

Filter design sifts deadly prions from blood supply

08/01/2004

BY STEVE SMITH

EAST HILLS, N.Y.—For the second time this summer, a major advance has been unveiled in the battle against infectious prions associated with Mad Cow Disease, Creutzfeldt-Jakob Disease (vCJD). The latest, announced last month by Pall Corp. (www.pall.com), is a proprietary filter technology that's said to reduce deadly prions from the blood supply before it is used for transfusions.

Earlier this summer, Serologicals Corp. (Atlanta, Ga.; www.serologicals) was granted a patent on its proprietary purification process that clears the Mad Cow prions from bovine-based pharmaceuticals. More than 60 percent of pharmaceuticals on the market have involved bovine-based products in their development or manufacture.

Pall Corp.'s Leukotrap affinity prion reduction filter is designed to offer a multi-targeted approach to blood safety by reducing leukocytes and infectious prions that are cell or non-cell associated. Tests indicate the filter reduces deadly Mad Cow Disease prions from the blood supply before it is used for transfusions.

Click here to enlarge image

Prion transmission via blood transfusion, though reduced in recent years, remains a concern for the public blood supply—particularly overseas. In December 2003, a case of vCJD was identified in a person who received a blood transfusion six years earlier from a donor who later died of the disease. In announcing its breakthrough to the International Society for Blood Transfusion annual meeting, Pall Corp. officials noted that since vCJD has an unknown and lengthy incubation period that is asymptomatic, there is no way to know how many have transmitted the disease via transfusion.

The Leukotrap affinity prion reduction filter, however, is designed to offer a multi-targeted approach to blood safety by reducing leukocytes and infectious prions that are cell or non-cell associated. According to the company, about 60 percent of prions in blood resides in cell-associated leukocytes and about 40 percent in non-cell associated plasma. Pall says its research indicates that the Leukotrap filter has an affinity to all types of prions, including aggregated, denatured and normal.

"We are moving this technology forward rapidly," reports Pall Corp. chairman and CEO Eric Krasnoff. "Blood centers and hospitals will soon be able to combine both prion and leukocyte reduction in a single, simple step."

Pall says it will also investigate broadening the technology to help identify Mad Cow Disease in cattle before it reaches the food supply.

With hopes of introducing the Leukotrap filter to hospitals by early next year, Pall says it expects that its technology will meet the requirements of the Council of Europe and will be ready for operational trials in Europe, where vCJD is most prevalent.

Because a majority of blood transfusions in the industrialized world is leukocyte-reduced, Pall is hopeful that its filtration development can swiftly fit into routine operating practice already in use in blood centers around the world.

Meanwhile, testing on the filter continues. With support from the New York Institute of Basic Research, Pall is studying the Leukotrap filter using Western blot assay, bioassay and animal models of prion disease to validate reduction of the infectious prions. Additional results on the animal model research are expected in the coming months.


Dobutamine cardiovascular magnetic resonance for the detection of myocardial ischemia with the use of myocardial tagging.
The purpose of this study was to assess the value of high-dose dobutamine cardiovascular magnetic resonance (CMR) with myocardial tagging for the detection of wall motion abnormalities as a measure of myocardial ischemia in patients with known or suspected coronary artery disease. Two hundred eleven consecutive patients with chest pain underwent dobutamine-CMR 4 days after antianginal medication was stopped. Dobutamine-CMR was performed at rest and during increasing doses of dobutamine. Cine-images were acquired during breath-hold with and without myocardial tagging at 3 short-axis levels. Regional wall motion was assessed in a 16-segment short-axis model. Patients with new wall motion abnormalities (NWMA) were examined by coronary angiography. Dobutamine-CMR was successfully performed in 194 patients. Dobutamine-CMR without tagging detected NWMA in 58 patients, whereas NWMA were detected in 68 patients with tagging (P=0.002, McNemar). Coronary angiography showed coronary artery disease in 65 (96%) of these 68 patients. All but 3 of the 65 patients needed revascularization. In the 112 patients with a negative dobutamine-CMR study, without baseline wall motion abnormalities, the cardiovascular occurrence-free survival rate was 98.2% during the mean follow-up period of 17.3 months (range, 7 to 31). Dobutamine-CMR with myocardial tagging detected more NWMA compared with dobutamine-CMR without tagging and reliably separated patients with a normal life expectancy from those at increased risk of major adverse cardiac events.


Q:

Using an IP-Address in URL to HTML Linked Resources (CSS, ...)

A well-known technique is to link resources (CSS, JavaScript, ...) with a separate DNS name for various reasons. Like this:
GET http://stackoverflow.com/
GET http://cdn.sstatic.net/stackoverflow/all.css

(Two different domain names)
Instead:
GET ...
GET http://92.60.242.2/stackoverflow/all.css

(One DNS lookup)
This means that two DNS lookups are required. Couldn't we just use an IP address instead of cdn.sstatic.net in order to save one DNS lookup?
Please assume that it is possible to use an IP host from the point of view of the server. Assume, that there is a dedicated resource serving server with a dedicated IP.

A:

Content delivery networks usually employ some sort of load balancing, often implemented at the DNS level (e.g. the name cdn.sstatic.net resolves to different IP addresses, based on the geographical location of the requester). Hard-coding the IP address would be counter-productive, as the request will always go to the same server (which might still be a load balancer in front of several backend servers, but all of them will be in the same location).


Q:

A simple C++ framework for Win32 Windows Applications?

Is there a simple/small framework (Other than .NET) which allows you to create windowed applications with C++ under Win32. Just like a little DLL I can include with my app.
It should have basic functions like creating a window , buttons , text edits and handling them.

A:

WTL is a set of lightweight templates that make writing Win32 windowing code quite easy (to the extend C++/Win32 can be easy).

A:

I would recommand Qt. It's an intuitive and user friendly framework. In addition it is cross platform if one day you want to deploy your app anywhere else.
Qt can be used through Visual Studio or through the QtCreator IDE installed with the framework (as well as QtDesigner [GUI editor]).

A:

Take a look at Win32++


In this case, Plaintiff has not provided any evidence which shows that when MERS assigned the mortgage to Plaintiff, it also transferred the interest in the underlying note. According to the mortgage assignment contract, MERS held legal title to the mortgage. There is no language in the agreement which transfers interest in the note to MERS. Because MERS did not hold title to the underlying note, it could not transfer any rights to the underlying note when it assigned the mortgage to Plaintiff. See LPP Mortgage Ltd v. Sabine Properties No. 103648/10,2010 N.Y. Misc. LEXIS 4216, at*7 (Sup. Ct. N.Y. Co. Sept. 1, 2010); Lamy , 824 N.Y.S.2d at 769 (Sup. Ct. Suffolk Co. 2006); HSBC Bank USA v. Miller, 26 Misc. 3d 407,411,889 N.Y.S.2d 430,433 (Sup. Ct. Sullivan Co. 2009). Without a transfer of title to the underlying note, Plaintiff cannot foreclose on the property based on default payment and lacks standing under CPLR 5 321 1 (a)(3)

[…]

Therefore, it is
ORDERED that Defendant’s motion to dismiss is granted; and it is further
ORDERED that this action is dismissed; and it is further
ORDERED that the clerk of the court directed to enter judgment accordingly.

NY SUPREME COURT: FINAL DISPOSITION

Here, there are no allegations or evidence that MERS was the owner of the note such that it could assign it to LPP. Thus, the assignment from MERS was insufficient to confer ownership of the note to LPP and it has no standing to bring this action. Kluge v. F umz ~1, 45 AD2d at 538 (holding that the assignment of a mortgage without transfer of the debt is a nullity); Johnson v. Melnikoff, 20 Misc3d 1142(A), “2 (Sup Ct Kings Co. 2008), n. 2, afr, 65 AD3d 519 (2d Dept 20 1 Oj(noting that assignments by MERS which did not include the underlying debt were a legal nullity); m e Elect ro pic Registration Svstem v, Coakley, 41 AD3d 674 (2d Dept 2007)(holding that MERS had standing to bring foreclosure proceeding based on evidence that MERS was the lawful holder of the promissory note and the mortgage).

Thus, even assuming arguendo that the language of the assignment from MERS to LPP could be interpreted as purporting to assign not only the mortgage but also the note, such assignment is invalid since based on the record, MERS lacked an ownership interest in the note. $ee LaSalle Bank Nat. Ass’n v. Lamv, 12 Misc3d 1191(A), “3 (Sup Ct Suffolk Co. 2006) (noting that “the mortgage is merely an incident of and collateral security for the debt and an assignment of the mortgage does not pass ownership of the debt itself ’);