Infectious bronchitis (IB) has an influential economic impact on the poultry industry, causing huge losses each year due to the condemnation of infected chickens. Despite the use of many kinds of vaccines in Iraq, it is common to find IB problems in vaccinated chickens. Information about the strains that affect Iraqi chickens is very limited. Therefore, we aimed to detect the currently circulating strains of IB virus that cause frequent outbreaks in egg layers despite the use of vaccination against the virus.Isolate detection, sequencing, and phylogenetic analysis were performed using a rapid IB virus antigen kit , flinders technology associates (FTA) card , and partial gene sequencing (16 positive FTA samples).The isolated strain was different from other strains, especially the strain isolated in the North of Iraq (Sulemania Strain) and shares 98% homology with an Israeli strain .Although more studies are needed to detect IB virus strains circulating in Iraq, this work lays the foundation for making a good strategy to control the disease and selecting vaccines that should be used in farms. Coronaviridae and the genus Gamma-coronavirus. The single-stranded RNA  of this enveloped virus has several open reading frames [An important viral disease affecting the poultry industry globally and locally in Iraq is the infectious bronchitis virus (IBV), which causes an acute, highly contagious respiratory disease. The identification of endemic IBV strains promotes better control of the disease and prevents production losses . IBV belg frames . AlthougIBV usually is subjected to changes due to mutations and genetic recombination. Therefore, a mutation in the S1 gene may affect virus tissue tropism and virulence . ConsequTo the best of our knowledge, only one study to date reports the molecular characterization of IBV that resulted in a broiler strain (Sul/01/09), identified in the Kurdistan region of Iraq. Therefore, the current report describes the role of IBV during an outbreak of the respiratory disease in an egg-layer farm in the Baghdad region of Iraq, investigates the genetic characteristics of this field strain by analyzing the S1 gene and compares it with other isolates registered globally for developing significant vaccines to control this disease.This study does not require ethical approval. However, all birds in the current work were treated humanely following International and National criteria of animal care and use.An outbreak occurred during January 2018 in Al-Janoob Company, located in the Al-Wahda district of Baghdad, Iraq. This was composed of eight caged houses of egg layers with a total number of 640,000 hens and 170,000 rearing pullets. The outbreak was not reported to the national authority. All flocks were vaccinated with commercial live-attenuated NOBILIS IB 4-91. Respiratory symptoms began in 28-day-old birds of an ISA Brown flock composed of 88,000 birds per house. Clinically, the symptoms were suspected to be IBV infections. Necropsies were performed, and gross lesions were evaluated.Sixty-four tracheal swabs (two swabs per bird) were collected during January 2018, from birds that showed such respiratory manifestations.Thirty-two tracheal swabs were used for serological diagnosis of IBV antigen using a Rapid IBV Ag Test Kit , which is a chromatographic immunoassay for qualitative IBV antigenic detection in avian swab samples. Tests were performed according to the manufacturer’s instructions.® FTA® card technology) with four sample areas per card  containing chemicals for lysing cells, denaturing proteins, and protecting nucleic acids against damage from nucleases, ultraviolet radiation, and oxidation [Samples were pooled on flinders technology associates (FTA) cards , where the extraction of the IBV RNA was performed using Kylt® IB-aCoV, Kylt® IBV-Variant 02, Kylt® IBV-Variant 4/91 (793b), Kylt® IBV-Variant Arkansas, Kylt® IBV-Variant D1466, Kylt® IBV-Variant D274, Kylt® IBV-Variant Italy02, Kylt® IBV-Variant Massachusetts, Kylt® IBV-Variant Q1, Kylt® IBV-Variant QX, and Kylt® IBV-IB80 (AniCon Labor GmbH). RT-PCR, CFX96, and CFX384  systems were used according to the following conditions: 50°C for 10 min and 95°C for 1 min , then 42 cycles of 95°C for 10 s, 60°C for 1 min, and read.RT-PCR runs were generated by AniCon Labor GmbH. Briefly, species-specific and variant-specific RT-PCR methods were performed to detect avian coronavirus  and IBV variants. Hybridization probe-based chemistry was used with the following primers: KyltThe amplified RT-PCR products were sequenced by AniCon Labor GmbH. Sequences of the Iraqi-IBV-strain S1 gene were deposited in the GenBank database available from the National Center for Biotechnology Information (NCBI) website to collect accession numbers.https://www.dnastar.com/), and Molecular Evolutionary Genetics Analysis (MEGA) X software (https://www.megasoftware.net/) were used to construct the phylogenetic tree.IBV S1 gene sequences obtained herein were compared with sequences of IBV deposited globally in the GenBank database using the NCBI-based BLAST search. The identities of the sequences were analyzed by DNAstar software  disease, and the mortality rates reached 8% for approximately 10 days. Birds suffered the following symptoms: Coughing, sneezing, and rales with depression. Postmortem findings included tracheal congestion, and caseous materials that partially obstructed the trachea and the tracheal bifurcations, and pneumonia and fibrinous airsacculitis . KidneysThirty-two (100%) of the examined samples were positive for the presence of the IBV antigen.Viral RNA from the FTA cards (four spots pooled) with the sample number (A1800428.001) showed positive results with species-specific and IBV-variant-specific RT-qPCR .A 630-bp and 730-bp fragments of the S1 protein-coding gene were sequenced by AniCon Labor GmbH. The sequences were deposited in the NCBI GenBank under accession numbers MH747093 and MH747094 with the name aCoV strain IQ1 and IQ2 spike glycoprotein (S1) gene, respectively.The genetic relationship between the S1 gene sequence of the MH747094.1 IBV-IQ2 strain and sequences of vaccine and other virulent strains are presented in IB has an influential economic impact on the poultry industry, causing huge losses each year due to the condemnation of infected chickens . Since Iet al. [et al. [The current work represents the first identifying and genotyping report of IBV that resembles the Israeli isolate in Iraq. The results of the sequencing and phylogenetic tree analysis indicate that our isolate shares 98% homology with the Israel variant 2 (IS 1494). In 2011, Zana  [et al.  also regTherefore, our work lays the main foundation including RNA-based S1 protein transcript sequencing and the related phylogenetic analysis, to initiate launching strategies for control of IBV in the field.Despite the intensive vaccination program in Iraq, IB outbreaks have occurred for many decades. In this study, we isolated a strain of IBV very different from that of local North Iraq (Sulemania Strain) but similar to an Israeli strain . Genetic characterization of the Iraqi circulating IBV strains is critical because of inadequate data from this location. More studies revealing such strains will pave the way for a good strategy of controlling the infections and understanding or developing types of vaccines that should be used in the future.AOA and HMA visited the infected farm, collected the samples, and performed the rapid IBV antigen detection. FA and YIK visited the infected farm, collected the samples, run the rapid IBV antigen detection, confirmed the results using FTA card that was sent to Germany, deposited the genetic information into the NCBI database, analyzed the differences between the strains of the virus, and drafted the manuscript. All authors revised the manuscript. All authors have read and approved the final manuscript.


Protease-activated receptor-2 (PAR2) has been implicated in multiple pathophysiologies but drug discovery is challenging due to low small molecule tractability and a complex activation mechanism. Here we report the pharmacological profiling of a potent new agonist, suggested by molecular modelling to bind in the putative orthosteric site, and two novel PAR2 antagonists with distinctly different mechanisms of inhibition. We identify coupling between different PAR2 binding sites. One antagonist is a competitive inhibitor that binds to the orthosteric site, while a second antagonist is a negative allosteric modulator that binds at a remote site. The allosteric modulator shows probe dependence, more effectively inhibiting peptide than protease activation of PAR2 signalling. Importantly, both antagonists are active in vivo, inhibiting PAR2 agonist-induced acute paw inflammation in rats and preventing activation of mast cells and neutrophils. These results highlight two distinct mechanisms of inhibition that potentially could be targeted for future development of drugs that modulate PAR2. Kennedy et al. report the pharmacological and in vivo profiling of two small molecule PAR2 inhibitors and an agonist. They conclude that while the small molecule agonist and one of the inhibitors bind to the orthosteric PAR2 binding site, the other inhibitor is a negative allosteric modulator, highlighting two distinct mechanisms of inhibition that could be targeted for future development of drugs that modulate PAR2. Protease-activated receptor-2 (PAR2) is predominately activated by serine proteases, such as trypsin and tryptase, which cleave between R35–S36 thereby exposing a new N-terminal sequence SLIGKV–, that acts as a tethered agonist for intramolecular PAR2 activation5. The activated receptor stimulates multiple different G protein-dependent and independent signalling pathways. PAR2 has been shown to signal through Gα proteins  across different cell environments, as well as through G protein-independent proteins including β-arrestins 1/27. In addition, synthetic peptide agonists corresponding to the tethered ligand sequence, either human SLIGKV-NH2 or the rodent sequence SLIGRL-NH2, can activate these signalling pathways via PAR210.Protease-activated receptors (PARs) are a unique family of G protein-coupled receptors with an unusual activation mechanism. Endogenously, the N terminus of these receptors is cleaved by extracellular proteases to reveal a tethered ligand sequence which can intramolecularly bind and activate the receptor11, cancer13, metabolic disease, both obestity14 and cardiovascular16, as well as in inflammation and inflammatory diseases18. The wide range of effects of PAR2 underscores the importance of this receptor in human physiology and disease. As a consequence, this target is highly sought after in drug discovery and for a number of years has been a focus of major pharmaceutical endeavours19. However, as yet the discovery of an effective drug has proved challenging for PAR2. The only marketed drug for a PAR is vorapaxar, a selective antagonist of PAR1 that is an antiplatelet treatment for improving restricted blood flow20. Even though PAR2 has close sequence homology with PAR1, the discovery of PAR2 antagonists has been less successful, with weak antagonists that only inhibit selected signalling pathways (e.g. GB88)22, or that show agonist properties in some cell types (e.g. C391)23. Development of tool compounds is important for better understanding of the mechanisms of PAR2 activation on different cell and tissue types and in diseases where PAR2 is a key mediator and potential therapeutic target.PAR2 has been shown to have roles in pain and migraine24. AZ8838 was derived from an initial high-throughput screen hit24 and AZ3451 was obtained from a DNA-encoded library technology screen, which also found an agonist, ‘compound 1’25. AZ8838 binds in an occluded pocket made up of transmembrane helices (TM) 1–3, 7 and extracellular loop 2 (ECL2), whereas AZ3451 occupies a pocket that faces the lipid bilayer and is formed by TM 2, 3 and 4. However, the definition of the orthosteric site of the tethered ligand remains elusive due to the lack of an agonist-bound PAR2 crystal structure. More recently, with the AZ8838-bound structure as a starting point, we used an extensive combinatorial approach of site-directed mutagenesis and computational modelling to propose the putative orthosteric site26.In 2017, we reported the crystal structures of PAR2 bound to AZ8838 and AZ34512 but is a more potent activator of multiple PAR2-dependent signalling pathways. Antagonists AZ8838 and AZ3451 can inhibit both G protein-dependent and independent pathways via PAR2 in vitro and exert anti-inflammatory effects in vivo in a rat model of PAR2 agonist-induced paw oedema. Our approach to interrogate ligand-receptor interactions, using molecular modelling and surface plasmon resonance in combination with functional assays, characterises AZ8838 as a competitive antagonist, whilst AZ3451 is a negative allosteric modulator. Our findings highlight coupling between ligand binding sites in the PAR2 receptor and illustrate opportunities for both orthosteric and allosteric inhibitors of PAR2 functions in vitro and in vivo.Here, we present the pharmacological characterisation of a novel agonist of PAR2 (AZ2429) and two novel antagonists (AZ8838 and AZ3451). Agonist AZ2429 is proposed to bind at the same site in PAR2 as the activating peptide SLIGKV-NH24, two novel antagonist series were discovered  signalling, relative to the most potent analogue, AZ3451. The imidazole series was developed from an initial weak high-throughput screen hit24. Optimisation resulted in compound AZ8838, although a constrained heterocycle (AZ0107) also maintained similar potency  PAR2 receptorred Fig. . For theays Fig. . Substit2 to Chinese hamster ovary cells transfected with human PAR2 (CHO-hPAR2). All compounds tested bound to human PAR2 (Table Ki = 6.9 ± 0.2) bound with higher affinity than the imidazoles (AZ8838 pKi = 5.2 ± 0.1). In agreement, both series bound to HEKexpi293F membranes expressing hPAR2 (AZ3451 pKi = 7.9 ± 0.1 and AZ8838 pKi = 6.4 ± 0.1) in competition binding assays against 3H-acetylated-GB110. Throughout this manuscript, the reference agonists GB110, 2f-LIGRLO-NH2 and SLIGRL-NH2 are interchanged as they have each been shown to bind at a common or overlapping site within PAR2 and are reported to activate the receptor in a similar manner27.The binding affinities of each series were compared for competitive binding with a europium-tagged fluorescent derivative of the known PAR2 agonist 2f-LIGRLO-NHR2 Table ; the ben2 resulted in stimulation of the Gq pathway triggering the Ca2+ mobilisation and inositol phosphate (IP1) signalling cascade. Inhibition of Ca2+ flux and accumulation of IP1 was evaluated in 1321N1 cells stably expressing human PAR2 (1321N1-hPAR2) using FLIPR and IP1 HTRF assays respectively. The compounds were potent antagonists against SLIGRL-NH2 in the Ca2+ assay with the benzimidazole series (AZ3451 pIC50 = 8.6 ± 0.1) more potent than the imidazole series  in an IP1 assay; the other two diastereomers were less potent .From DNA-encoded library screening, one agonist series was identified429 Fig.  was iden3H]-GB110. AZ2429 was able to displace the probe  with a 7-fold higher potency than SLIGKV-NH2 (pEC50 = 5.9 ± 0.1) and a 6-fold lower potency than peptide mimetic GB110 (pEC50 = 7.5 ± 0.1)  and was 120-fold more potent than SLIGKV-NH2  Fig. . No Ca2+2 in PAR226. The underlying assumption was that AZ2429 and the DNA-encoded compound (DNA-AZ2429) bind to the receptor in a similar way. Docking calculations were therefore guided to bury the benzyltriazole deep into the receptor pocket leaving the methylamide facing the extracellular region to allow for the attachment of the DNA-tag, which was present when this compound was identified25.To investigate where the novel agonist AZ2429 binds in PAR2, molecular docking simulations were performed using the AZ8838-bound crystal structure of PAR2. Modelling placed the agonist AZ2429 in the previously reported binding site for SLIGKV-NH2. Towards the extracellular domain, the aminotetralin–carboxamide is positioned around the proposed binding region of the third hydrophobic residue of SLIGKV-NH2 fulfilling the prerequisite that the terminal amide of AZ2429 is freely accessible from the opening of the receptor pocket.Docking predicts that the heterocycle of AZ2429 adopts a T-shape orientation towards H227, between polar residues D228 and Y82. This is the part of the binding pocket predicted to bind the N-terminal serine of the tethered ligand and which binds the imidazole of AZ8838 in the crystal structure, although the triazole of AZ2429 is slightly closer to D228 Fig. . The ben24, revealing slow binding kinetics and a corresponding residence time of about 84 min. In contrast, binding of the antagonist AZ3451 was characterised by high affinity with accompanying rapid association kinetics to the receptor and a residence time of about 2 min  into the hind paws of Wistar rats caused an acute oedema manifested by paw swelling that peaked after 30 min and subsided within 4 h. PAR2 antagonists, AZ3451 (10 mg kg−1 s.c. 30 min prior) and AZ8838 (10 mg kg−1 p.o. 2 h prior) were evaluated for their capacity to inhibit this agonist-induced paw inflammation when administered prior to agonist and both showed 60% reduction of paw swelling at the doses given . The identification of PAR2 antagonists that inhibit G protein-dependent and -independent signalling pathways in vitro and show anti-inflammatory activity in vivo in rodents provides two new tool compounds for interrogating PAR2 functions.Previous studies illustrate the challenges associated with the development of PAR2 antagonists. High millimolar concentrations of N1-3-methylbutyryl-N4-6-aminohexanoyl-piperazine (ENMD-1068) were required to dose-dependently attenuate joint inflammation2 peptide agonist. Molecular modelling predicted that AZ2429 binds in the same pocket as SLIGKV-NH226.AZ2429, a novel agonist, discovered through a DNA-encoded library screen and identified after chiral separation, activates G protein-dependent and -independent signalling pathways via PAR2. With a similar potency to peptidomimetic GB110, this small molecule is more potent than the SLIGKV-NH26. Interestingly, agonist AZ2429 was able to bind, thought to be because of its small molecule nature and due to its heterocycle binding in a stable T-shape orientation between H227, D228 and Y82. This allowed for the first biophysical characterisation of interactions between the two distinct antagonist binding sites (AZ8838 and AZ3451) and the agonist binding site of PAR2. With this new tool, and supported by pharmacological characterisation in vitro, we have identified two distinct functional mechanisms for inhibition of PAR2.Remarkably, unlike other PAR2 agonists, AZ2429 can bind to the PAR2 stabilised receptor (StaR), which contains several mutations to improve its thermostability. The mutations lock the receptor in an inactive-like configuration, closing off the agonist binding site such that the peptide/tethered ligand agonists are unable to bind2 building on a previous study26 which indicated key receptor residues necessary for peptide-induced receptor activation. Importantly, we confirm that AZ8838 is also a competitive antagonist of the endogenous tethered ligand, suggesting that it may bind at the same site in PAR2 as the tethered ligand.In SPR studies, when AZ8838 was bound to the receptor, AZ2429 was no longer able to bind, consistent with overlapping binding sites. The predicted docking pose of AZ2429 agrees with the SPR data and visualises how AZ8838 may bind in a subpocket of the agonist binding site. We show that AZ8838 is a competitive antagonist of 2f-LIGRL-NH33, AZ3451 could restrict inter-helical conformational rearrangements. Thal et al. suggest that upon binding to its site between TM 2 and 4, AZ3451 could prevent the rotation and movement of TM3 required for receptor activation. This would render the receptor in a more stable ground state conformation which could explain the significant increase in residence time of AZ2429. Pharmacological analysis of the antagonist activity of AZ3451, in the presence of peptide or protease agonists of PAR2, supports the SPR binding studies; indicating that this antagonist is non-competitive (Schild slope ≪ 1.0) and a negative allosteric modulator (α < 1). The data are consistent with it causing reduced agonist binding to PAR2 and the decreased association rate of binding of AZ2429 in the presence of AZ3451.In contrast, SPR studies identified allosteric coupling between the more remote binding sites of AZ3451 and AZ2429. AZ2429 had altered binding kinetics in the presence of AZ3451 and vice versa. The agonist may help stabilise intermediate active conformation(s) of PAR2 that are distinct from the ground state of the receptor, which could lead to the observed reduction in residence time and thus decrease in binding for the antagonist. Like other extrahelical antagonistsMoreover, SPR studies showed that both antagonists could bind simultaneously to PAR2, as would be expected with the well-separated binding sites revealed in the protein crystal structures. The almost unchanged binding kinetics of AZ3451 in the presence of AZ8838 suggest that their binding sites are not conformationally linked, although it is noteworthy that the affinity is reduced.By combining molecular modelling, SPR and in vitro pharmacological characterisation, we present a newfound understanding of the interaction between binding sites in PAR2. At the putative orthosteric site, located between TM1, 3, 6, 7 and ECL2, binding of the tethered ligand, peptide or small molecule (AZ2429) agonists causes receptor activation. AZ8838 appears to bind at a site overlapping this orthosteric site to inhibit receptor signalling in a competitive manner. AZ3451 binds to a more remote site on the outside of the 7TM helical bundle and inhibits receptor signalling from this allosteric site through bidirectional, long-range coupling to the orthosteric site.50 = 8.5 and 6), despite it being well-characterised that protease activation of PAR2 is monophasic34. Therefore, AZ3451 exhibits probe dependence, an intriguing characteristic of allosteric antagonists35, which has previously been characterised on muscarinic receptor subtypes36. The biphasic effect was independent of assay format, cell line and receptor expression level; being observed in three different cell lines, 1321N1-hPAR2 and CHO-hPAR2 stably expressing human PAR2 and HT-29 cells with endogenous levels of PAR2, and two different signalling pathways (Ca2+ and ERK1/2). The biphasic response was not perturbed by a PAR1 antagonist, suggesting it was not due to interaction with PAR1. Mutagenesis around the known AZ3451 binding site appeared to disrupt the biphasic response. AZ3451 inhibition of trypsin-induced PAR2 activation at mutant receptors [F154A and G157C] was monophasic with a lower affinity (pIC50 = 6), suggesting that AZ3451 may have a second lower affinity binding site in PAR2. Although a second binding site for AZ3451 was not previously found, it is perhaps because the StaR conformation does not allow binding to the second site, or the second site is only accessible upon proteolytic cleavage of the N terminus. A number of studies have highlighted differences in activation of PAR2-mediated signalling by the tethered ligand compared to peptides38. It is possible that the movement of the tethered ligand into the binding site induces further conformational changes in the receptor, which might have a profound effect on the outside of the helical bundle where the high-affinity binding site of AZ3451 is located, explaining why AZ3451 induces partial inhibition of only the tethered ligand-activated state. A recent paper reported a similar finding, a PAR2 antagonist attached to a PEG linker partially inhibited SLIGKV-induced activation of Ca2+ signalling via PAR2, with IC50 = 0.1 µM, however, had no effect on trypsin-induced activation of PAR239. Antagonists that can distinguish between peptide and protease-induced activation of PAR2 provide opportunities to improve our understanding of the endogenous mechanism of activation by proteases.Allosteric antagonist, AZ3451 displayed intriguing pharmacology. In binding experiments against labelled peptidomimetic agonists, there was monophasic competition, and this was consistent with the inhibition of peptide-induced activation of PAR2 signalling. However, AZ3451 inhibition of the activation of PAR2 signalling by trypsin was biphasic -NH2 was synthesised according to Hoffman et al.40. [3H-GB110] was synthesised as in Cheng et al.24. Recombinant human trypsin (referred to as trypsin) was from Polymum Scientific. Chemical synthetic routes of compounds and analytical data are described in supplementary methods.SLIGKV-NH2 were performed as previously described38. CHO-hPAR2 cells were seeded overnight in 384-well plate at 2.5 × 104 cells/well and blocked with 2% BSA for 60 min. Cells were simultaneously exposed to both Eu-tagged-2f-LIGRLO(dpta)-NH2 (300 nM) and test compounds (AZ3451 or AZ8838) for 15 min. Cells were washed 3× with PBS supplemented with 20 µM EDTA, 0.01% Tween and 0.2% BSA followed by addition of DELFIA Enhancement Solution (Perkin Elmer) for 90 min. Time-resolved fluorescence was measured with PHERAstar FS reader (BMG Labtech).Competition binding experiments against Eu-tagged-2f-LIGRLO(dtpa)-NH3H]-GB110 were carried out on membranes isolated from HEKexpi293F cells expressing human PAR2. In 96-well plates, at final assay concentration stated in parenthesis, protein (30 µg per well), label (25 nM), and test compounds AZ3451, AZ8838, AZ2429 (0.6 nM–100 µM), SLIGKV-NH2 (0.3 nM–55 µM) or unlabelled GB110 (0.3 nM–10 µM) were incubated for 2 h at room temperature with shaking. The reaction was then filtered through 0.5% PEI-coated filter plates using a BiomekFX. The filter plate was then dried for 1 h at 50 °C. Scintillation fluid was added to each well, before reading the plate in a Microbeta reader.Competition binding experiments against radiolabel  using FLIPR TETRA (Molecular Devices) to detect agonism. For each concentration, the difference between the maximum peak Ca2+ response and baseline response is reported. Evaluation of antagonism was performed after an additional 30 min incubation at room temperature followed by addition of SLIGRL-NH2 (2 µM), trypsin (39 nM) or tryptase (1 µM) at EC80 for activation of PAR2 and detection of Ca2+ flux. Antagonism experiments were also carried out on 1321N1-PAR2[WT], 1321N1-PAR2[F154A], and 1321N1-PAR2[G157C] as described. Experiments with trypsin in 1321N1-hPAR2 cells were performed in the presence of 1 µM Vorapaxar (Chemtronica) to remove any interference of PAR1 activity. Inhibition of Ca2+ release on rat PAR2 was measured in the same way using 10 000 KNRK cells per well seeded in poly-D-lysine coated 384-well plates (Greiner), 30 min preincubation with the compound before activation of PAR2 with EC80 of SLIGRL-NH2 (120 µM) and real-time detection of Ca2+. Antagonism experiments for CHO-hPAR2 and HT-29 cells were performed in 96-well plates at 30,000 cells/well. Cells were loaded with Fluo-3 AM calcium dye (Sigma-Aldrich) for 60 min at 37 °C in HBSS with 1% FBS, 0.2% pluronic acid, 20 mM HEPES and 2.5 mM probenecid prior to the addition of antagonist. The antagonist was pre-incubated for 60 min at 37 °C followed by the addition of bovine trypsin (30 nM) at EC80. Intracellular [Ca2+] flux was measured using FLIPR TETRA (Molecular Devices) and the difference between the maximum peak Ca2+ response and baseline response is reported.For evaluation of Caq HTRF assay kit (Cisbio). Compounds were pre-incubated with 15 000 cells per well in white small-volume 384-well plates (Greiner) for 30 min at 37 °C in 20 mM HEPES with HBSS. Next, EC80 of SLIGRL-NH2 (70 µM) supplemented with 50 mM LiCl was added and incubated for 60 min at 37 °C. Detection of IP1 production was performed according to the manufacturer’s protocol and HTRF was measured using PHERAstar (BMG LabTech). Data were normalised to [IP1] using a standard curve. Activation of PAR2 and subsequent IP1 production was measured in a similar way, but with 60 min compound incubation in the presence of 50 mM LiCl before the detection of IP1.Inhibition of PAR2-induced inositol-1-phosphate (IP1) production was measured in 1321N1-hPAR2 cells using IP-One G2. Antagonist compounds (30 µM–1 nM) were incubated with the cells for 30 min prior to the addition of agonist SLIGRL-NH2 (10 µM) at EC80 and further incubation for 90 min at 37 °C. Working detection solution (12 µl) was then added to each well, incubated at room temperature in the dark for 60 min and the chemiluminescence signal read on the EnVision microplate reader (Perkin Elmer).β‐Arrestin-2 recruitment was measured in U2OS-hPAR2 cells using the DiscoverX assay kit, according to the manufacturer’s instructions. Briefly, cells were seeded in McCoy’s 5A media containing 10% FBS at 4000 cells per well in 384-well plates and incubated overnight at 37 °C, 5% CO2. Antagonist compounds (30 µM–1 nM) were incubated with the cells for 30 min prior to the addition of agonist SLIGRL-NH2 (10 µM) at EC80 and further incubation for 5 min at 37 °C. The supernatant was removed from the wells and lysis buffer was added to the cells and incubated with shaking at room temperature for 30 min. The cell lysate was transferred into a small-volume 384-well plate containing detection antibodies and incubated for 4 h at room temperature in the dark. Plates were read for HTRF signal on PHERAstar (BMG LabTech). In addition, phosphorylation of ERK1/2 was measured in CHO-hPAR2 using AlphaLISA SureFire Ultra pERK1/2 (Thr202/Tyr204) (Perkin Elmer). Cells were seeded at a density of 2000 cells per well in a 384-well proxiplate and incubated overnight. Antagonists were pre-incubated for 30 min at 37 °C prior to the addition of agonist (1 µM 2f-LIGRL-NH2 or 50 nM trypsin) for 10 min at 37 °C. After incubation, cell lysis buffer was added with shaking for 30 min. Antibodies and beads were added and incubated for 4 h at room temperature. AlphaLISA signal was measured using a PHERAstar (BMG LabTech).Phosphorylation of ERK1/2 was measured in U2OS-hPAR2 cells using Phospho-ERK1/2 (Thr202/Tyr204) assay kit (64ERKPEH Cisbio) and the 2 plate manufacturer’s protocol with the following modifications. Cells were seeded in McCoy’s 5A media containing 10% FBS at 15,000 cells per well in 384-well plates and incubated overnight at 37 °C, 5% COi model in GraphPad Prism 8 (GraphPad Software) to determine binding affinity. Pharmacology in vitro potency data was analysed with a 4-parameter logistic fit using the equation y = Bottom + (Top − Bottom)/(1 + 10)^((LogXC50-x)*(HillSlope)) where y is the response, x is the 10-logarithm of the drug concentration, Bottom is no response and Top is full response in GraphPad Prism 8 or Genedata Screener (Genedata). pEC50 and pIC50 data are presented as mean ± standard error of the mean (s.e.m.) derived from the number of independent experiments stated. Schild slope analysis was calculated using Graphpad Prism 8 Gaddum/Schild EC50 global fitting  competitive model. The most rigorous method for quantifying agonist-antagonist interactions is to globally fit the Gaddum/Shild model to all agonist dose–response curves41. Schild slope quantifies the shifts in agonist response in the presence of antagonist, a Schild slope of 1 indicates the antagonist is competitive and extra-sum-of-squares F-test (p < 0.05) was used to compare the calculated Schild slope against a hypothetical Schild slope of 1. Allosteric interaction studies between ligands in the calcium flux assay were fitted to the following operational model of allosterism42:Em is the maximum possible cellular response and held constant at 100, [A] and [B] are the concentrations of the orthosteric and allosteric ligands, respectively, n represents the slope of the transducer function and was held constant at 1. KA and KB are equilibrium dissociation constant of the orthosteric and allosteric ligands respectively as determined with Eu-tagged-2f-LIGRLO(dtpa)-NH2, pKA of 2f-LIGRLO-NH2 = 6.1, pKB of AZ3451 = 6.9. Parameters τA and τB denote the capacity of orthosteric and allosteric ligands, respectively, to exhibit agonism, logτA = 0.7 and logτB = 0 since AZ3451 showed no agonist activity. β represents the efficacy factor between orthosteric and allosteric ligands. α represents the cooperativity factor between orthosteric and allosteric ligands . Allosterism for AZ3451 in the calcium flux assay was determined and analysed. The relative activity was calculated using (EmaxEC50’)/(Emax’EC50) with the Hill slope help constant at 1. EC50’ and Emax’ denote the EC50 and Emax in the absence of allosteric modulator, while EC50 and Emax denote those measured in the presence of allosteric modulator. When the Hill slope = 1, the relative activity plot approaches an asymptote corresponding to the estimate of the γ value.Binding data were analysed using the One site – Fit K43. Docking of AZ2429 was performed using Glide SP44 to a refined PAR2 model previously described26. In short, the PAR2 complex structure 5NDD24 was refined to fit peptide ligands and peptidomimetics by placing GB88 in the proposed binding site and creating a new model using the 5NDD structure as a template in Prime45 to allow for side-chain rearrangements. Ligand placement during docking was guided using a positional constraint of a phenyl moiety in place of the phenyl ring of AZ8838 from the PAR2 complex structure. Positional constraints were used with a tolerance of 2.4 Å.AZ2429 was prepared using Ligprep−1. The NTA surface was chosen to allow full control of the steric orientation of the tethered protein and was conditioned by a one-minute injection of a 350 mM EDTA solution (pH 8.3) followed by a one-minute injection of running buffer supplemented with 0.5 mM NiCl2. To allow for a covalent tethering after the initial capture step, the conditioned surface was activated for 7 min with 50 mM NHS and 200 mM EDC. This was immediately followed by an injection of PAR2 in running buffer at a concentration of 80–120 µg ml−1 with a contact time of 5–10 min to achieve the desired densities of 8000–9000 RU. The deactivation of residual esters was achieved through a waiting period of 4–5 h that also resulted in a more stable SPR signal. Reference surfaces were prepared accordingly, omitting the injection of protein over the activated reference surface. The subsequent binding experiments were all performed at a flow rate of 30 µl min−1 and by employing the method of single-cycle kinetics. This approach involves the sequential injection of a compound concentration series without regeneration steps. A contact time of 60 s was selected, which was followed by a 30 min dissociation phase to allow for a proper estimation of the dissociation rate constant. Compounds were dissolved in DMSO to 10 mM and a digital dispenser HP D300 (Tecan) was used to set up the compound concentration series using 8 concentrations with a 2-fold dilution pattern. The tested concentrations for AZ3451 and AZ2429 have been 8, 16, 32, 64, 128, 256, 512, 1024 nM and 75, 150, 300, 600, 1200, 2400, 4800, 9600 nM, respectively. Prior to the analysis of compound binding, three running buffer blanks were injected to equilibrate the instrument. The data collection rate was set to 10 Hz and all experiments were repeated at least four times to allow for error estimations. Due to the low DMSO mismatch (maximum 0.1%) introduced by the compound addition, no solvent correction was required. To evaluate compound binding to the PAR-AZ8838 complex, the antagonist AZ8838 was supplemented into the running buffer at a concentration of 50 µM and the instrument was equilibrated for 1 h before performing the single-cycle kinetic study as described above. Similarly, binding of AZ2429 to the PAR-AZ3451 complex was investigated after addition of 1 µM AZ3451 to the running buffer followed by 1 h equilibrium period, while the addition of 10 µM AZ2429 to the running buffer was required to ensure >99% saturation for single-cycle kinetic study of AZ3451 binding to the PAR2-AZ2429 complex.The SPR binding experiments were performed on a Biacore S200 optical biosensor unit  at 30 °C. Sensor chips Series S NTA  were equilibrated at room temperature prior to use. The running buffer for protein tethering and subsequent ligand binding experiments was 50 mM HEPES, 250 mM NaCl, 0.1% (w/v) LMNG, 0.005% (w/v) CHS, 0.025% (w/v) CHAPS, pH 7.4, if not stated otherwise. The tethering of PAR2 was performed at a flow rate of 5 µl min46 in order to extract kinetic and affinity data. All data were expressed as mean ± s.e.m. and further analysed using GraphPad Prism 8. Statistical differences were assessed using student’s t-tests (two-tailed) for data sets of two and one-way ANOVA for data sets of three. Significance was set at +p < 0.05, ++p < 0.01, +++p < 0.001, ++++p < 0.0001 compared to AZ3451, and *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to AZ2429.Prior to the fitting of the data, the reference-subtracted data was further analysed by subtracting a similar single-cycle kinetic experiment containing only buffer injections in order to correct for injection artefacts, systematic noise and instrument drift. This double-referenced data has been fitted using a simple 1:1 interaction model as described by Karlsson et al.2 at 350 µg/100 µl saline/paw. Saline only was given to sham paws. Treated animals received AZ3451  or AZ8838  30 min or 2 h prior to administering 2f-LIGRLO-NH2, respectively. Control animals received either DMSO s.c. or 20% DMSO/80% olive oil via oral gavage. All injected paws were measured  with a digital calliper  just before 2f-LIGRLO-NH2 or saline administration (0 h), as well as at 0.5, 1, 2 and 4 h post-oedema induction. Data were expressed as a normalised % change in cross-sectional area (mm2) from the baseline against maximal swelling at 30 min induced by 2f-LIGRLO-NH2 alone. At the 30 min time point, a portion of animals was euthanised by CO2 inhalation and their paws collected for further analyses. All in vivo experimental procedures and animal handling were conducted with approval from the Animal Ethics Committee at the University of Queensland which follows NHMRC and ARRIVE guidelines. After experimentation, animals are humanely euthanized by CO2 inhalation as stipulated by the ethical agreements.Male Wister rats  were bred at the Animal Resources Centre . Following Australian ethical standards, animals were air transported to the Institute for Bioengineering and Nanotechnology . Animals were housed at room temperature and kept in 12 h light/dark cycles, with standard chow and water provided ad libitum, for at least a 48 h acclimatisation period before experiments. Hind paw swelling was induced by i.pl. injection of 2f-LIGRLO-NHRat paw tissues were collected, fixed in 10% neutral-buffered formalin (Sigma-Aldrich) for 2 h at 4 °C, transferred into falcon tubes containing 25% sucrose in PBS and left overnight at 4 °C. Tissues were then embedded with Optimal Cutting Temperature compound  and stored at −80 °C before making cryo-sections for histological analysis. Frozen sections (5 µm) were made using a cryostat-microtome . For mast cell staining, slides were briefly washed with distilled water before staining with 0.1% toluidine blue  for 3 min. Slides were then dehydrated and mounted in DPX mountant . For immunohistochemistry, slides were briefly rinsed with PBS to remove OCT. Except for tryptase staining, antigen retrieval was performed by incubating slides in citrate buffer  at 95 °C for 20 min. They were then cooled down briefly and incubated with blocking medium  for 1 h at room temperature in a humidity chamber. Samples were incubated with primary antibody medium  overnight at 4 °C. The next day, samples were washed with PBS and incubated with the secondary antibody medium  for 2 h at room temperature. For tryptase staining, slides were incubated with primary antibody medium (as above with tryptase antibody at 1:100 dilution) for 4 h, then the secondary antibody medium for 2 h. Finally, slides were dried, counterstained with DAPI , and sealed with clear nail polish. Primary antibodies for tryptase , histamine , neutrophil elastase  and ED1  were purchased from commercial sources. Secondary detection antibody was purchased from Abcam, Australia. All microscopic images were obtained using an Olympus BX-51 upright microscope with Olympus DP-71 12Mp colour camera, utilising DP Capture and DP Manager software packages . Tryptase-, neutrophil elastase- and ED1-positive cells were quantified using FIJI/ImageJ 1.42q software, USA.2, and stored at −80 °C. Samples were cut with a scalpel, weighed, and added to MPO buffer  to a concentration of 200 mg ml−1, before homogenising using zirconium oxide beads  and a Bullet Blender® homogeniser . Homogenised tissues were diluted to 100 mg ml−1 with MPO buffer containing 1% hexadecyl trimethylammonium bromide . Samples were then sonicated for 5 min, centrifuged at 13,000 × g for 10 min at 4 °C, and the clear supernatant for each sample was collected. For MPO release, supernatant (30 μl) was added to 200 μl of MPO buffer containing 0.167 mg ml−1o-dianisidine HCl (Sigma-Aldrich USA) and 0.001% H2O2 in a clear 96-well plate, which was read immediately at 460 nm every min for 20 min .Rat paw tissues were collected, snap-frozen with liquid Nt-tests (two-tailed) for data sets of two, one-way ANOVA for data sets of three or more with Bonferroni post-hoc tests, or two-way repeated-measures ANOVA with Tukey post-hoc tests for temporal data sets, as appropriate. Significance was set at +p < 0.05, ++p < 0.01, +++p < 0.001, ++++p < 0.0001 compared to sham, and *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to disease control.Experimental results were expressed as mean ± s.e.m. Graphs were plotted and data analysed using GraphPad Prism7. Statistical differences were assessed using Student’s Pharmacological data are presented as mean ± s.e.m. of the indicated number of replicates stated in the relevant figure legend or data table. Graphs show individual data points (from at least two replicates) with error bars denoting s.e.m. Replicates are defined as independent repeats of the experimental protocol. Statistical analyses were conducted as outlined in the relevant method section.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Supplementary FilesSupplementary Data 1Reporting Summary


The emissions of several semivolatile compounds families such as polycyclic aromatic hydrocarbons (PAHs), polychlorobenzenes (ClBzs), and polychlorophenols (ClPhs), with special interest in the emissions of the most toxic compounds, i.e., polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (dl-PCBs), were analyzed. A notable decrease in PCDD/F and dl-PCB formation was achieved in most of the experiments, especially for those runs performed under an oxygen-rich atmosphere (λ = 0.50), where the addition of sludge was beneficial with inhibition ratios Ri ≥ 0.25. An inhibition ratio of 0.75 showed the best results with almost a 100% reduction in PCDD/F formation and a 95% reduction in dl-PCB formation.Persistent organic pollutant inhibition in the combustion process of polyvinyl chloride (PVC) by prior addition of an inhibitor is currently being studied, reducing the emission of pollutants, and thus reducing the large amount of waste PVC destined for landfill. In this work, the use of sewage sludge (SS) as an alternative to chemical inhibitors to improve the quality emissions of the incineration of polyvinyl chloride waste (PVC e-waste) was studied and optimized. Different combustion runs were carried out at 850 °C in a laboratory tubular reactor, varying both the molar ratio R Polyvinyl chloride (PVC) e-waste is a highly chlorinated thermoplastic whose generation has significantly increased in the last years and its disposal involves nowadays a substantial environmental uncertainty . In factA simple scheme of the process occurring during thermal decomposition of wastes is shown in At present, many studies have been performed to reduce the emission of PCDD/Fs formed from the thermal treatment of waste with different methods, such as photocatalytic decomposition, catalytic oxidation, and through ozone application ,7,8. HowMost studies focus on the use of specific nitrogen- and sulfur-based compounds , obtaining good results of inhibition in PCDD/F formation and even reducing also NOx emissions ,12,13. CIn addition, the possibility of using waste rich in nitrogen and/or sulfur as a source of inhibitory compounds is getting more and more relevance because of the reduced pollutant emission factor, besides reducing waste accumulation in landfills. In this way, the effect of adding sludge from urban wastewater treatment plants (WWTPs), which have a high content of nitrogen compounds, has been specially studied in three different ways: By direct mix of the waste , by usinSewage sludge (SS) is a semi-solid waste that is generated in urban WWTPs as a consequence of water treatment processes. The use of SS is normally linked to obtaining compost for its use in agriculture as main destination. However, the large amounts generated and the fact that not all the SS can be devoted to its use as compost due to its pollutant load, makes its use as an alternative fuel be the main option. Moreover, it is worth mentioning that during the compost production process from this waste, PCDD/Fs could be generated in certain situations ,20,21. ATherefore, given the nitrogen and sulfur content of SS and high chlorine content of PVC e-waste, this work aims to study the potential of combining both by direct mixture in a co-incineration process to reduce the emission of PCDD/Fs, PCBs and other organic micropollutants which are incomplete combustion products. The study results could be applied in thermal treatment units such as incinerators, blast furnaces and furnaces of cement and ceramics production. In this way, it could help in reducing the amount of pollutants produced, too.In addition, this work is in line with the sustainable development goals (SDGs) 3, 9 and 11, stablished in the 2030 agenda for sustainable development by the United Nations, which aim for a pollution-free planet. Environmental degradation is responsible for nearly one in four deaths (12.6 million people a year), as well as a variety of health problems and widespread destruction of vital ecosystems, and in particular air pollution, which has been estimated to cause about 6.5 million deaths per year .Electrical wires used in this study were supplied by General Cable, Co . These wires were composed by a PVC cover and a cross-linked polyethylene (PE) insulator with copper as a conductor, already used in previous works ,25,26,27Nevertheless, in the present work only the plastic fraction formed by the cover and the insulation material were used, consisting of e-waste of PVC (82.8%) with impurities of PE (17.2%). This plastic fraction was manually separated from the metallic fraction and then crushed using a cutting mill  until obtaining a particle size of less than 1 mm, small enough to guarantee the homogeneity of the samples in the runs.This e-waste was previously characterized employing an elemental microanalyzer (Thermo Finnigan Flash 1112 Series) to furnish total content (wt%) of carbon, hydrogen, nitrogen and sulfur, and an X-ray fluorescence spectrometer  to provide the rest of elements . Table 1SS were provided by Cemex España SA  and prior to their use, SS were dried in an oven at 105 °C for 24 h and reduced to dust by a mixer. The ash content was measured following the UNE-EN-14775:2009  at 550 °The different experiments performed to determine the different compounds produced during the combustion of PVC and sludge were carried out in a horizontal reactor with a combined displacer-furnace system.The reactor has been used in previous works ,26 and cTo introduce the sample into the combustion zone, a linear actuator  with a movement capacity up to 500 mm and speeds between 0.1 and 800 mm/s was used. The reproducibility of the actuator (±0.02 mm/s) ensured a homogeneous feed flow. The actuator was coupled to a controller that allows the creation of different movement programs, by defining the initial and final points, as well as the speeds associated with the trajectories between programmed points. This equipment allows the sample to move in parallel towards the combustion zone in the same direction as the air flow, with a maximum flow rate of 500 mL/min.i) was defined according to the Equation (1):i = 0.25, Ri = 0.50, and Ri = 0.75 were considered.The mixtures of both wastes were made following a “molar inhibition ratio” that relates the amount of nitrogen and sulfur contained in the sludge, with the amount of chlorine contained in PVC e-waste. The molar inhibition ratio . Oxygen ratio (λ) was calculated based on the synthetic air supplied to the experiment with the Equation (2):mbustion ,33.−4 and 1.6·× 10−3 m/s, as well as the air flow rate with values between 400 and 589 mL/min. Values of λ = 0.15, and λ = 0.50 were considered.In this way, the oxygen ratio would be λ = 1 for a combustion process where the oxygen present is the stoichiometric one and λ = 0 for a reaction with total absence of oxygen. For each experiment, the oxygen ratio was adjusted by changing the speed of the samples entering the combustion chamber with values between 6 ×·10Once synthetic air flow rate (measured at 1 atm and 20 °C) and horizontal furnace temperature were attained and constant (850 °C in every run performed), the boats were introduced in parallel with air flow at constant speed with the appropriate sample load. Before each experiment, a control run with no sample loading was carried out using the same experimental conditions (blank).i = 0.25, Ri = 0.50 and Ri = 0.75). For every mixture, experiments were carried out under two atmospheres with different amounts of oxygen (λ = 0.15 and 0.50).The whole study was performed with a total of eleven samples, including blank experiments. Apart from PVC e-waste and sewage sludge, the samples consisted of 3 mixtures of sewage sludge and PVC e-waste in different proportions of (S + N)/Cl . For the extraction of the samples, pure toluene was used as solvent, since the extraction of all the compounds was done together and toluene must be used to extract the heaviest compounds. Once the extract was obtained, it was separated into two fractions. A first fraction with 30% of the total weight of the extract was used for the analysis of PAHs, ClBzs, and ClPhs, and a second fraction with the remaining 70% of the extract was used for the analysis of PCDD/Fs and PCBs. Both extract fractions were concentrated on a Büchi model R210/V rotary evaporator until a final volume of around 1 mL.The extraction process of the resin samples was performed in an ASE2 steam in a multiple Pasvial evaporator until a volume of approximately 1.5 mL was reached prior to the analysis by high-performance gas chromatography (Agilent 6890N) with mass spectrometry (Agilent 5973N) (HRGC-MS) as explained elsewhere [10 with a concentration of 2000 µg/mL in dichloromethane  was added as a recovery standard. The addition of a known quantity of this standard allows to calculate the recovery percentage of the internal standards throughout the process.Fraction 1 samples were transferred to a chromatography vial to proceed to the concentration with a Nlsewhere . Three µTM automatic cleaning equipment, from the company FMS, which is an automated fluid management system, capable of automatically purifying extracts from samples of different nature for the subsequent analysis of compounds toxic at trace levels. This system uses three different disposable columns supplied by the manufacturer: An acid-base multilayer silica column, an alumina column, and an activated carbon column. PAHs, phenols, acids, and esters, as well as oils and lipids, are retained on the silica column. The basic alumina column allows to separate PCDD/Fs from other organic compounds and also retains lipids and phenols, separating the PCB fraction (both non-ortho and mono-ortho). Finally, the activated carbon column allows the PCDD/Fs to be isolated from other organic compounds, as they are retained at the top of the column due to their planar configuration, being subsequently eluted in reverse flow.Fraction 2 samples, for the analysis of PCDD/Fs and PCBs, were purified using a Power-Prepv/v). Both fractions were concentrated in the rotary evaporator to an approximate volume of 1 mL. Next, 50 µL of nonane was added and the sample concentration was completed with the aid of a gentle stream of N2 to a final volume of about 50 µL. Just before the analysis, the 13C-isotopically labeled recovery standards were added to the different fractions (PCDD/Fs and PCBs), which allowed to calculate the percentage of recovery or loss of the internal standards throughout the extraction, purification and concentration process. Specifically, 10 μL of the EPA-1613ISS solution  for PCDD/Fs and 10 μL of the WP-ISS solution diluted to 200 ppm (Wellington Laboratories) for PCBs were employed.After purification, two fractions were obtained: One containing the PCDD/Fs in toluene and the other containing the PCBs in a 50% mixture of dichloromethane/hexane  coupled to triple quadrupole mass spectrometry (Agilent 7010B) (GC-MS/QQQ). The system is also equipped with an Agilent 7693A automatic injector. The methods use Multiple Reaction Monitoring (MRM) mode for data acquisition. For each target, two specific precursor ions as well as two corresponding product ions and collision energies were selected: One MRM transition for quantitation and one for qualification. The quantification of PCDD/Fs and PCBs was carried out by isotope dilution methodology, which uses the relative response factor for each congener with respect to the corresponding labeled standard, following the requirements of the U.S. EPA method 1613  for diox12 for an Ri of 0.75, which was slightly higher. For λ = 0.50, the recoveries were between 40% and 100%.In order to check that the results obtained were reliable, the recovery of the deuterated standards for each sample was verified. The results obtained for the recoveries of the deuterated standards in all the experiments performed at λ = 0.15 were around 100%, with the exception of the recovery value of chrysene-dThe results obtained for the 16 priority PAHs were determined using the isotope dilution technique and are presented in i = 0.50 and Ri = 0.75, as could be expected even in the case of no inhibition action for the mixture of both residues. However, for Ri = 0.25 the total concentration of PAHs was even slightly above the values reached for the pure PVC e-waste sample.The decomposition with a λ = 0.15 presented much higher concentrations of PAHs than runs at λ = 0.50, as expected due to a less efficient combustion . Values i = 0.50; this is due to the presence of sufficient oxygen in the atmosphere to remove a large part of the hydrocarbons formed in the pyrolytic stages.For λ = 0.50, the highest concentrations of PAHs were obtained for PVC e-waste and the lowest concentrations were obtained for sludge. This time, lower intermediate concentrations of PAHs were obtained for the mixtures, even when an increase in the inhibition ratio was not directly correlated with a decrease in the concentration of PAHs, as observed for λ = 0.15. In this case, the lowest concentration of PAHs was obtained for a RPVC e-waste contained around 30 times more chlorine than sludge see , so a hiIn order to be more accurate and to determine if the sludge acted effectively as an inhibitor in their formation, the concentration of PAHs that should have been obtained (expected) in the samples PVC/sludge simply considering their independent contributions was calculated based on the sludge and PVC e-waste fractions of each mixture. The results of the comparisons of the expected theoretical concentrations and those actually obtained are presented in The percentages of reduction in the formation of PAHs were calculated for the different inhibition ratios and for both oxygen ratios λ = 0.15 a and λ =2+ ions.As can be seen in i = 0.50, the concentration of undesirable compounds also increased in most of the cases and in the total amount of PAHs emitted. However, this increase in PAHs formation went from being around 30% for Ri = 0.25 to 15% for Ri = 0.50. Therefore, there was a general inhibition, compared to the experiment carried out with an Ri = 0.25.For RFor an inhibition ratio of 0.75, a significant reduction in the concentration of PAHs was obtained with respect to the expected theoretical amount, producing a significant inhibition of between 20 and 40%, with the only exceptions of naphthalene and acenaphthylene. Based on the experiments performed, it could be concluded that in order to obtain a significant reduction in some of the PAHs formed when working in an atmosphere very poor in oxygen (λ = 0.15), the best inhibition ratio was 0.75, but it would be recommended not to co-incinerate both wastes as naphthalene, acenaphthylene and phenanthrene are precisely the major compounds emitted and are the least inhibited.The reduction in the concentration of pollutants at a low inhibition ratio begins to be noticeable when working with higher oxygen ratios. It can be seen in As the inhibition ratio increased, the concentration of PAHs present in the sample decreased notably, some of the compounds being reduced by more than 80% b.i of 0.75, a high percentage of inhibition between 60 and 80% was observed for every compound, reaching 100% for dibenzanthracene. For Ri = 0.50 a greater reduction was observed in the lightest PAHs, which were in turn the most abundant. Therefore, although for Ri = 0.75 a greater reduction was obtained on average in all PAHs, at a global level the results obtained for Ri = 0.50 were better.For an Ri = 0.50 and Ri = 0.25 the mixtures that showed the best and worst results, respectively.As a conclusion, λ = 0.50 was always better to reduce the emission of PAHs, so in the presence of more oxygen, the formation of PAHs is lower, being RThe analysis of the results obtained from the combustion of the samples for the formation of these compounds is presented jointly, since the analysis methods were similar, and both were measured in the mass spectrometer in SIR mode.13C-labeled standards for all the experiment runs in ClBz analysis were around 100%, except for the first two 13C-labeled standards, whose recoveries were around 20%. As these two were more volatile, their losses were more likely in the evaporation phase.The results obtained for the recoveries of the 13C-labeled standards were between 40 and 80% for all the runs, with the exception of the last standard whose recovery was around 15%. This could be due to the fact that it is a heavier and more thermolabile compound, which can degrade further on the chromatographic column during GC-MS analysis.For the ClPhs, the recoveries of the i). The results at λ = 0.15 do not show a great difference when varying inhibition ratios. For those compounds appearing with the highest concentrations, it was observed that the best inhibition ratio was 0.75. Clearly, monochlorobenzene was the isomer obtained with the highest yields, as usual in thermal treatments.i were those which contained more chlorine and, therefore, facilitate the formation of these compounds.The results of ClBzs emissions at λ = 0.50 barely showed any difference between the inhibition ratios of 0.50 and 0.75. However, the worst results were clearly obtained for the inhibition ratio of 0.25, most probably due to the fact that the samples with a lower RThe best oxygen ratio for every inhibition ratio was undoubtedly λ = 0.50, where the concentration of the compounds was approximately 6 times lower for all cases, facilitated by a predominance of decomposition mechanisms due to the higher amount of oxygen present during the thermal treatment.In In the same way as for ClBzs, the best oxygen ratio for the mixtures with inhibition ratios 0.25 and 0.50 was λ = 0.50.13C-labeled dioxin standards in all the experiments performed at both oxygen ratios were close to 100%, except for some runs at λ = 0.15 which were between 80% and 90%. However, all values were well within the ranges allowed by the US EPA method 1613.The results obtained for the recoveries of the i = 0.25. However, for the hexa-, hepta- and octachlorinated congeners, the mixture Ri = 0.25 presented higher dioxin emissions. The explanation of this phenomenon could be related to the composition of the sludge, which is shown in 3·6H2O, FeCl2·4H2O, Fe2O3, CuCl2·2H2O, and CuCl2·2H2O, which could already exist in the sample previously or be produced during the combustion process.The results obtained at different inhibition ratios and oxygen ratios are presented in The lowest emissions in the case of the mixtures for λ = 0.15 were obtained for an Ri of 0.75 and the highest emissions for Ri = 0.25. For an inhibition ratio of 0.25, the obtained concentrations were much higher than for the other ratios, being this sample the one that contained the most amount of chlorine of all the mixtures tested, in addition to having small amounts of metals (iron and copper) that seemed to have catalyzed the formation reactions of PCDD/Fs.For λ = 0.50 (greater presence of oxygen), the effect of the presence of dioxin formation catalysts in the mixtures was less evident, and what happened at λ = 0.15 when the formation of PCDD/Fs in the mixtures exceeded that of the PVC sample was not observed. Here it is important to remark the high PCDD/Fs emissions for the PVC e-waste alone at λ = 0.50 compared to the rest of the experiments.Observing the results for different inhibition ratios at λ = 0.50, the lowest emissions were obtained for an inhibition ratio of 0.75, as in the case of λ = 0.15, being in general lower the levels of PCDD/Fs that were emitted.i = 0.25) or with high inhibition ratios (Ri = 0.75), λ = 0.50 was always better to reduce the concentration of PCDD/Fs. However, for intermediate inhibition ratios around 0.50, the emissions obtained were similar for both oxygen ratios.It can be observed that when working with small inhibition ratios , the addition of sludge hindered the process, causing an increase in the concentration of PCDD/Fs that were emitted, probably due to the catalytic effect of metals discussed in the previous section.i = 0.75 where practically all the congeners reached a 100% reduction. The worst inhibition ratio was 0.50, reaching reductions between 30 and 80%. In this way, the global inhibition efficiencies achieved were 89.2%, 71.4%, and 98.8% for the inhibition ratios 0.25, 0.50 and 0.75, respectively.For λ = 0.50, very promising results were obtained for every inhibition ratio studied, being the optimum ratio Ri = 0.75 was always better to minimize the emission of PCDD/Fs, so in the presence of more sludge, the formation of PCDD/Fs is much lower for both oxygen atmospheres, being λ = 0.50 the oxygen ratio that clearly showed the best results in terms of the inhibition efficiency.As a conclusion, R13C-labeled PCB standards for all the experiments performed with λ = 0.15 and λ = 0.50 were around 100%, with small variations, but within the limits allowed by the US EPA 1668 method for PCBs.The results obtained for the recoveries of the The results obtained for PCBs emissions are shown at different inhibition ratios and oxygen ratios, including pure materials, in i = 0.25 and Ri = 0.75, especially for an Ri = 0.25. Despite slight variations, it can be concluded that the lowest concentrations were obtained for an inhibition ratio of 0.50, which means that intermediate sludge-PVC e-waste mixtures were those with the greatest reduction in PCBs emissions. When working in an atmosphere very poor in oxygen, adding high amounts of sludge to the mix to try to inhibit the formation of PCBs should be avoided, because the opposite effect could occur. Consequently, it is crucial to determine the optimal mixture to ensure a reduction in the formation of PCBs, knowing that it lies between an inhibition ratio of 0.25 and 0.75.The emissions obtained from PCBs for λ = 0.15 and an inhibition ratio of 0.50 showed relatively small differences compared to sludge and PVC e-waste. However, the emissions were even higher for mixtures with Ri = 0.50 for the less chlorinated congeners. The increase in the emission of PCBs for λ = 0.50 with respect to λ = 0.15 was only noticeable for PVC e-waste emissions.When considering the concentrations obtained for PCB emissions for λ = 0.50, different trends were observed from those obtained for λ = 0.15. As can be seen in i = 0.75. It can be concluded that when working at higher oxygen ratios, the use of inhibition ratios higher than 0.50 would be beneficial.For λ = 0.50 the best inhibition results were obtained with RIncreasing the value of the oxygen ratio from 0.15 to 0.50 showed a variation in the results at different inhibition ratios. In this later case, it was observed that the worst inhibition ratio was 0.50, when for the case of λ = 0.15, it was the relation for which the best results were obtained.For all mixtures, an oxygen ratio λ = 0.50 was always better to reduce PCB emissions.Once the best inhibition ratios that should be used at each oxygen ratio were determined, the percentages of reduction of PCBs were calculated for the different inhibition ratios and oxygen ratios, as shown in For λ = 0.15, the results obtained in the study of the inhibition capacity of the sludge addition were unfavorable for all the mixtures tested, producing basically an increase of the emission of PCBs in all congeners and being the inhibition ratios of 0.25 and 0.50 the ones that provided the worst and best results, with an increase of 236.5% and 15.4% in total emissions of PCBs, respectively.For λ = 0.50, the results obtained were really favorable in all cases, obtaining a decrease in the concentration of PCBs for all mixtures and being the inhibition ratio of 0.75 the one that provided the best results reaching a global inhibition efficiency of 91.9%.It can be concluded that for all mixtures, an oxygen ratio λ = 0.50 was always better and even imperative to reduce PCB emissions.i), which refers to the proportion between the sum of nitrogen and sulfur content of sludge with respect to chlorine content of PVC e-waste, were studied. A total of 3 inhibition ratios were studied , following the evidence that sulfur and nitrogen compounds inhibit the formation of pollutants such as PCDD/Fs in combustion processes.In this work, samples of PVC e-waste, sludge, and mixtures with different inhibition ratios , which refers to the amount of oxygen available to react with the sample during its combustion. The objective was to evaluate how oxygen affected the formation of these pollutants. Two different oxygen ratios (λ = 0.15 and λ = 0.50) were considered, searching to recreate incomplete combustion conditions that can occur in these situations.Regarding PAHs, when working in poor oxygen conditions (λ = 0.15) the best results were obtained for an inhibition ratio of 0.75, reducing some of the compounds by up to 60% with respect to the expected theoretical formation. However, the best results were obtained for λ = 0.50, where there was a significant reduction in the formation of pollutants for all inhibition ratios, being 0.75 the best inhibition ratio.The concentrations obtained for ClBzs and ClPhs were in many cases near zero, especially for ClPhs . For these compounds, not all the factors mentioned above were analyzed due to the few values that could be obtained (most of the results being under the detection limit). However, it was enough to find a clear and visible trend. It was determined that for ClBzs in poor oxygen conditions (λ = 0.15), the best inhibition ratio was 0.50, while for higher oxygen ratios (λ = 0.50), the best inhibition ratio was 0.75. As for PAHs, the lowest ClBzs emissions were obtained when working at λ = 0.50. For ClPhs, the results obtained were similar to those obtained for ClBzs, but in this case for both oxygen ratios the best inhibition ratio was 0.75.Regarding the analysis of PCDD/F emissions, the best inhibition ratio was 0.75 for both oxygen ratio conditions. However, for poor oxygen conditions (λ = 0.15), all inhibition ratios caused an increase in the formation of PCDD/Fs with respect to the expected theoretical amount. On the other hand, for a higher oxygen ratio (λ = 0.50), a very significant reduction in the emission of all PCDD/Fs was observed for every mixture, being 0.75 the best inhibition ratio. In addition, the global inhibition efficiencies reached were 89.2%, 71.4%, and 98.8% for the inhibition ratios 0.25, 0.50, and 0.75, respectively.Finally, the results regarding dl-PCB emissions followed a similar trend to what was detected for all the previous analyzed compounds. When the emissions were produced with a λ = 0.15, the best inhibition ratio was 0.50. However, an increase in the formation of PCBs was observed for all inhibition ratios, with respect to the expected theoretical values. PCB emissions in a higher oxygen presence (λ = 0.50) offered the best results for an inhibition ratio of 0.75, too. In addition, there was a significant reduction of emissions for every inhibition ratio, between 60% and 95% of inhibition. Finally, the global inhibition efficiencies achieved were 86.7%, 76.5%, and 91.9% for the inhibition ratios 0.25, 0.50, and 0.75, respectively.The results of analyzed emissions were divided into 4 families of compounds: The first dedicated to the study of PAHs, the second to ClBzs and ClPhs, the third to PCDD/Fs and, finally, the fourth, dedicated to dl-PCBs. In order to select the best operating conditions for reducing the whole pollutant emissions, the main conclusions obtained in each block are summarized here:i ≥ 0.25, being 0.75 the inhibition ratio that delivered the best results in most cases.To conclude, the optimal conditions for a general inhibition effect in every pollutant studied were achieved under higher oxidative conditions (λ = 0.50). Moreover, the addition of sludge to PVC e-waste combustion was really convenient, working with inhibition ratios RThus, it would be interesting to study these emissions on further investigations for a molar inhibition ratio of 0.8, which correspond exactly to a mass ratio 1:1 , being able to simplify as much as possible the mixing process during its co-incineration and being beneficial for its possible future industrialization and inhibition capacity improvement.The results also revealed a great sensitivity to the amount of oxygen available during the reaction in sub-stochiometric conditions, so it will be crucial to have this parameter well controlled, especially in incinerators, where this element can be scarce in some points and incomplete reactions are frequent.


Viral vectored vaccines are desirable alternatives for conventional infectious bronchitis virus (IBV) vaccines. We have recently shown that a recombinant Newcastle disease virus (rNDV) strain LaSota expressing the spike (S) protein of IBV strain Mass-41 (rLaSota/IBV-S) was a promising vaccine candidate for IBV. Here we evaluated a novel chimeric rNDV/avian paramyxovirus serotype 2 (rNDV/APMV-2) as a vaccine vector against IBV. The rNDV/APMV-2 vector was chosen because it is much safer than the rNDV strain LaSota vector, particularly for young chicks and chicken embryos. In order to determine the effectiveness of this vector, a recombinant rNDV/APMV-2 expressing the S protein of IBV strain Mass-41 (rNDV/APMV-2/IBV-S) was constructed. The protective efficacy of this vector vaccine was compared to that of the rNDV vector vaccine. In one study, groups of one-day-old specific-pathogenic-free (SPF) chickens were immunized with rLaSota/IBV-S and rNDV/APMV-2/IBV-S and challenged four weeks later with the homologous highly virulent IBV strain Mass-41. In another study, groups of broiler chickens were single (at day one or three weeks of age) or prime-boost (prime at day one and boost at three weeks of age) immunized with rLaSota/IBV-S and/or rNDV-APMV-2/IBV-S. At weeks six of age, chickens were challenged with a highly virulent IBV strain Mass-41. Our challenge study showed that novel rNDV/APMV-2/IBV-S provided similar protection as rLaSota/IBV-S in SPF chickens. However, compared to prime-boost immunization of chickens with chimeric rNDV/APMV-2, rLaSota/IBV-S and/or a live IBV vaccine, single immunization of chickens with rLaSota/IBV-S, or live IBV vaccine provided better protection against IBV. In conclusion, we have developed the novel rNDV/APMV-2 vector expressing S protein of IBV that can be a safer vaccine against IB in chickens. Our results also suggest a single immunization with a LaSota vectored IBV vaccine candidate provides better protection than prime-boost immunization regimens. Infectious bronchitis (IB) is a highly contagious viral disease of poultry . It is pCoronaviridae. The genome of IBV is a non-segmented, positive-strand RNA of about 27.6 kilobase (kb) in length. The genome consists of five basic and four accessory genes in the order 5′-leader-replicase-UTR-S(spike)-3a,3b-M(membrane)-E(envelope)-5a,5b-N(neuclocapsid)-UTR-polyA-3′. IBV is an enveloped virus and contains S, M, E, and N proteins [Infectious bronchitis virus (IBV) is a member of the family proteins .Vaccination is the most efficacious approach to control IB. Currently, both inactivated and live attenuated vaccines are used to control IB . HoweverParamyxoviridae and have a non-segmented, negative-sense RNA genome that contains six genes in the order 3′-N-P-M-F-H-L-5′ [Viral-vectored vaccines provide an alternative approach for current IBV vaccines. Although several viral vectors have been used to develop novel IBV vaccines, each viral vector has its own advantages and disadvantages ,16,17,18F-H-L-5′ . To dateF-H-L-5′ . APMV-1 F-H-L-5′ . These sF-H-L-5′ . in ovo immunization and administration in presence of pre-existing immunity to APMV-1. We have recently shown that a recombinant lentogenic APMV-1 strain LaSota expressing the S protein of IBV was a promising vaccine candidate against IBV . HoweverIn this study, we have evaluated the chimeric rNDV/APMV-2 vector virus as a vaccine vector against IBV in comparison with the rAPMV-1 strain LaSota (rLaSota) vector and a modified version of the rAPMV-1 strain LaSota (rLaSota-527) vector, with increased replication . We gene50). This virus stock was used to infect chickens in IBV challenge experiments.Human epidermoid carcinoma (HEp-2) cells and chicken embryo fibroblast (DF1) cells were obtained from the American Type Culture Collection  to recover recombinant viruses by reverse genetics and for in vitro characterization of recovered viruses, respectively. The cells were grown in Dulbecco’s minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS). Recombinant viruses were propagated in allantoic fluids of 10-day-old embryonated specific pathogen free (SPF) chicken eggs . The virulent IBV strain Mass-41 was propagated in 10-day-old embryonated SPF chicken eggs. The allantoic fluid containing IBV was harvested five days after infection, centrifuged at low speed to sediment debris and the supernatants were aliquoted and stored at −70 °C. The titer of the stored virus was determined by 50% embryo infectious dose  of phosphoprotein (P) gene of NDV, one T nucleotide as intergenic sequence (IGS), gene start (GS), Kozak, open reading frame (ORF) of the S gene of IBV and PmeI restriction enzyme site. The ORF of S gene of IBV strain Mass-41 was codon optimized for higher expression in chicken cells . The correct sequence of flanking region was confirmed by sequence analysis using a forward primer from the P gene of NDV at upstream of the PmeI restriction enzyme site and a reverse primer from the M gene of NDV at downstream of the PmeI restriction enzyme site. The rNDV-APMV-2 or rLaSota-527 expressing S protein of IBV were recovered from cDNAs using reverse genetics as described previously [A plasmid BR322 containing full anti-genomic cDNA of NDV strain Beaudette C (BC) in which the F and HN ectodomains of NDV were replaced with corresponded F and HN ectodomain of APMV-2 (GenBank Accession No. EU338414) was constructed previously . A plasmeviously . The receviously . In two The expression of S protein by rNDV-APMV-2/IBV-S and rLaSota-527/IBV-S in comparison with the expression of S protein by rLaSota/IBV-S was assessed by Western blot. The monolayer of DF1 cells was infected with rLaSota, rLaSota/IBV-S, rLaSota-527/IBV-S or rNDV-APMV-2/IBV-S in the presence of 10% allantoic fluid. The DF1 cell lysates at almost similar level of infections were collected 30 h after infection. The expression of S protein in cell lysates was detected by Western blot analysis using a chicken polyclonal anti IBV serum.6 EID50/bird, in 200 µl volume, of rNDV-APMV-2/IBV-S and rLaSota/IBV-S through oculonasal route, respectively. The chicks of Group 3 were inoculated with 200 µl PBS through oculonasal route, and chicks of Group 4 were left uninfected. Four weeks after immunization, all birds of groups one to three were infected with 104 EID50/bird, in 200 µl volume, of virulent IBV strain Mass-41 through eye drop route and birds of group four were left uninfected. The severity scores of clinical signs of IBV including, nasal discharge, ocular discharge, and difficulty in breathing  were recorded twice a day for 10 days post-challenge. At day five post-challenge, tracheal swab samples were collected from all chickens and placed into 3 mL cold DMEM containing 10X antibiotics. The viral RNA was quantified in swab samples by an IBV-N gene-specific RT-qPCR using the protocol described previously [A total of 34 one-day-old SPF chicks obtained from Charles Rivers (Charles Rivers MA) were divided into three groups (Groups 1–3) of ten chicks and one group of four chicks (Group 4). The chicks of Groups 1 and 2 were immunized with 10eviously .7 HAU/bird, in 200 μL volume of rNDV-APMV-2/IBV-S, rLaSota/IBV-S expressing S protein, and/or recommended dose 200 μL in volume of a live attenuated Mass-type IBV vaccine by the oculonasal route based on the immunization regimen for each group listed in 5.5 EID50/bird of virulent IBV strain Mass-41 by the oculonasal route. The severity scores of clinical signs of IBV were recorded and post-challenge tracheal viral load were evaluated as described for IBV challenge experiment in SPF chickens. A total of 48 one-day-old broiler chickens were divided into eight groups, six in each. In a single or prime-boost immunization (prime at day one and boost at week three of age), chicks were immunized with 2All animal experiments were performed in our USDA approved Biosafety level-2 and Biosafety level-2 plus facilities following the guidelines and approval of the Institutional of Animal Care and Use Committee (IACUC), University of Maryland.t-test was used to compare two groups. To avoid bias, IBV protective experiments were designed as blinded studies.Data were analyzed among groups using a one-way-ANOVA test. The The transcription cassette containing the S gene of IBV was inserted between the P and M genes in two individual plasmids containing cDNA of the full length antigenomic RNA of NDV-APMV-2 or LaSota-527. rNDV-APMV-2 expressing IBV S protein (rNDV-APMV-2/IBV-S) and rLaSota-527 expressing IBV S protein (rLaSota-527/IBV-S) were recovered from cDNAs successfully. A rLaSota expressing S protein of IBV (rLaSota/IBV-S) which we generated previously also was used in the in vitro and in vivo experiments of this study . The recThe expression of S protein of IBV by rNDV-APMV-2/IBV-S, rLaSota-527/IBV-S, and rLaSota/IBV-S was detected by Western blot. Like rLaSota/IBV-S, rNDV-APMV-2/IBV-S and rLaSota-527/IBV-S expressed the S protein at high level . For the6 EID50/bird of rNDV-APMV-2/IBV-S or rLaSota/IBV-S and four weeks post-vaccination they were challenged with 104EID50/bird.In order to evaluate the comparative protective efficacies of rNDV-APMV-2/IBV-S and rLaSota/IBV-S against IBV, one-day-old SPF chicks were immunized with 10Compared to non-immunized chicks, both rNDV-APMV-2/IBV-S and rLaSota/IBV-S provided significant and comparable protection against clinical signs of IBV A. Howeve5.5 EID50/bird of virulent IBV. The results showed that immunization of chickens with rLaSota/IBV-S, rNDV-APMV-2/IBV-S, or live IBV vaccine, no matter which regimens of immunization, reduced the post-challenge IBV clinical signs significantly. However, compared to other groups, immunization of broiler chickens with rLaSota/IBV-S or live IBV vaccine at week three of age provided better protection of clinical signs of IBV. Single immunization of chickens with rLaSota/IBV-S or live IBV vaccine at week three of age reduced post-challenge tracheal viral load significantly, whereas single immunization of chickens at day one of age and prime-boost immunization regimens did not decreased post-challenge viral load  were presented in We also compared protective efficacies of rLaSota-527/IBV-S and rLaSota/IBV-S for protecting against clinical signs and in preventing post-challenge viral shedding against IBV challenge. Four-week-old chickens were immunized and three weeks after immunization, they were infected with virulent IBV strain Mass-41. The clinical signs of IBV were recorded, and post-challenge tracheal IBV RNA load was quantified by RT-qPCR. Three weeks after immunization the humoral antibodies induced against LaSota were detected by the HI assay. Our results showed that compared to rLaSota-527/IBV-S, rLaSota/IBV-S provided slightly better protection against clinical signs. Compared to rLaSota-527/IBV-S, the reduction in post-challenge tracheal viral load for rLaSota/IBV-S was 5 Login ovo vaccination. The control of IB is achieved by vaccination . HoweverOur result showed that rNDV/APMV-2/IBV-S did not develop plaques in DF1 cells, whereas, rLaSota/IBV-S and rLaSota-527/IBV-S formed a large size plaques. This result was not unexpected, because APMV-2 does not form syncytia or plaques in DF1 cells . A previImmunization of SPF chicks with rNDV-APMV-2/IBV-S and rLaSota/IBV-S provided comparable levels of protection against IBV clinical signs. However, in this IBV protective experiment, neither rLaSota/IBV-S nor rNDV-APMV-2/IBV-S decreased the post-challenge viral load, significantly. One possible explanation for this is that chicks at day one of age have an immature immune system when vaccinated for IBV ,38,39,40It was shown by Cavangah et al. that amino acids within hypervariable region (S1 subunit) of IBV spike proteins are responsible for major neutralizing epitopes . We prevSimilar to the live IBV vaccine, single immunization of broilers at three weeks of age with rLaSota/IBV-S significantly reduced post-challenge IBV genome in the trachea of chickens; however prime-boost immunization (heterologous or homologous regimens) with rLaSota/IBV-S and/or rNDV-APMV-2/IBV-S did not reduce post-challenge tracheal IBV RNA load at six weeks of age. These results suggest that not only double vaccination with rAPMVs/IBV-S did not improve the challenge outcomes, but, surprisingly, it increased the post-challenge clinical signs and tracheal viral load. We also observed that compared to single immunization, prime-boost immunization of SPF chickens with a live IBV vaccine provided less protection (unpublished). This could be due to the high titer of antibodies induced against non-neutralizing epitopes within the S protein of IBV and/or due to the negative effect of the over infection with vaccine strains on the development of balanced immune responses against IBV. Previously, it was shown that coronavirus infections may cause antibody dependent enhancement (ADE) . A studySingle immunization of broiler chickens with rNDV-APMV-2/IBV-S or rLaSota/IBV-S at day one of age or at week three of age provided protection against a high dose of IBV challenge. However, compared to single immunization at day one of age, immunization of three-week-old broilers with rLaSota/IBV-S provided better outcomes, in protecting from disease and preventing viral shedding. These results suggest that consistent with our previous study  and a reFurthermore, our results showed that compared to rLaSota/IBV-S, immunization of SPF chickens with rLaSota-527/IBV-S, did not provide better protection, indicating that increased replication of the vector may not necessarily lead to increased protection.In conclusion, in this study we have shown that the novel chimeric rNDV/APMV-2 vector expressing S protein of IBV, which is a better candidate for IBV vaccination because it is safer for young chicks and chicken embryos, provided protection against IBV in chickens. Our results also suggest a single immunization with an APMV vectored IBV vaccine candidate, with optimal replication, provides better protection than prime-boost vaccination against IBV.


Introduction: Wrong site surgery (WSS) is a preventable error. When these events do occur, they are often devastating to the patient, nursing staff, surgeon, and facility where the surgery was performed. Despite the implementation of protocols and checklists to reduce the occurrence of WSS, the rates are estimated to be unchanged.Materials and Methods: An innovative technology was designed to prevent WSS through a systems-based approach. The StartBox Patient Safety System was utilized at six sites by 11 surgeons. The incidence of near misses and WSS was reviewed.Results: The StartBox System was utilized for 487 orthopedic procedures including Spine, Sports Medicine, Hand, and Joint Replacement. There were no occurrences of WSS events. Over the course of these procedures, medical staff recorded 17 near misses utilizing the StartBox System.Conclusions: StartBox successfully performed all tasks without technical errors and identified 17 near miss events. The use of this system resulted in the occurrence of zero wrong site surgeries. Wrong site surgery (WSS) continues to plague medical facilities across the globe despite implementation of initiatives, checklists, and protocols. WSS refers to surgery that is incorrectly performed on the wrong side, wrong spine level, wrong anatomy, wrong patient, or the wrong procedure. Estimates on the incidence of WSS vary widely, ranging from 0.09 to 4.5 per 10,000 procedures . This poWith the guidance of proper processes, checklists, and safeguards, the Joint Commission has declared that WSS is a preventable event that should never occur. In support of this expectation, the Joint Commission introduced a Universal Protocol in 2004 that provides guidelines for the fundamental elements of a WSS prevention protocol. The Universal Protocol includes requirements for marking of the surgical site, confirmation of patient identity, confirmation of the intended procedure, and review of these details among the surgical team during a time-out immediately prior to the start of surgery . While UThe StartBox Patient Safety System  is an innovative technology that was evaluated to assess its ability to prevent WSS. This evaluation was performed using cases performed by 11 surgeons at six sites with the StartBox System. The System collects procedure data including a description of the surgery, evaluation of any near misses or other safety benefits added to the case by StartBox, and postoperative evaluation of the occurrence of WSS events defined as any procedure performed at the wrong site including, incorrect procedures, and procedures performed on the wrong patient.Lavender, for Left; Rose, for Right; Neutral Gray, for No Laterality  and a data reporting tool. The software application of StartBox is an easy-to-implement, standardized platform that improves communication between the surgical team and the patient; between the surgeon's practice and surgical facility; and among care providers along the patient care continuum . The appterality . This cos injury . With thUpon completion of the procedure, the case data, including near misses, is stored and aggregated to generate predictive analytics related to future WSS prevention protocol improvements and training opportunities. Near misses related to WSS would include incorrectly booked surgery and improperly performed presurgical time-outs .The study was carried out with sequential series at each site using retrospective, deidentified data. The system is designed to protect the confidentiality, availability, and integrity of personal health information as required by HIPAA and satisfy the compliance requirements of institutional ethics committees.The StartBox System was utilized for 487 orthopedic procedures . The proNo Gos  No Gos were due to incorrect procedure information including site or description. Five (5) No Gos were due to laterality mismatch.Six (6) No Gos were recorded by the preop nurse at check-in on the day of surgery (9), followed by the circulating nurse in the OR (4) and the clinic scheduler (3). A surgeon recorded one No Go . The second was a spine case with a mismatched informed consent and dictated preoperative surgical plan . The patient identified the error after listening to the dictation at the hospital before being prepped for surgery and inquired with the nurse, who then confirmed with the surgeon. The informed consent was then corrected at preop. Both of these procedures were successfully completed with the StartBox System.Two final examples of Use of StartBox did not result in any reported impacts or use impedance to patient workflow at the clinic or hospital and there were no delays in surgery due to technical difficulties during the time-out or failure of the system to release a surgical blade after completion of the time-out.British psychologist James Reason suggested in his Swiss Cheese model of accident causation that catastrophic safety failures are almost never caused by isolated errors committed by individuals. Instead, most accidents result from multiple, smaller errors in environments (the holes in the cheese) with serious underlying system flaws. In this model, errors made by individuals result in disastrous consequences. Reason also emphasized that human error is inevitable, and that a systems approach can catch errors before they occur or block them from causing harm .The Hierarchy of Intervention Effectiveness, first introduced in 1999 by the Institute for Safe Medication Practices, presents a risk management theory that ranks intervention methods from least to most effective. Human-focused interventions such as education, training, rules, policies, and checklists are rated toward the bottom of its scale . While nConsidering this background, current measures fall short in two categories. First, WSS prevention protocols do not address potential sources of error, according to the Swiss Cheese model. Moreover, relying on a checklist without computerization or constraints is an inferior and generally acknowledged less-effective means of error prevention.With the end goal of improving WSS prevention protocols and ideally eliminating WSS altogether, the StartBox System was developed to enhance all three intervention methods. System-focused improvements include standardizing and streamlining workflows, as well as complementing existing electronic medical record systems. This system boosts human-oriented methods that contribute to effective communication, such as the huddle described in TeamSTEPPS (Team Strategies and Tools to Enhance Performance and Patient Safety)  as well The goal of this study is to report the early experience using this innovative system comprising the recording of the decision for surgery, verification of the procedure record for all the constituents in the patient care continuum, confirmation of procedure accuracy during the surgery time out, and the use of a physical forcing function before the surgical incision.In the clinical evaluation that is the focus of this report, StartBox was utilized with 487 procedures to standardize a process that is intended to prevent WSS. Users reported a good experience using the system, including the anecdotal feedback summarized in Near misses, sometimes referred to as close calls or potential adverse events, are defined as acts of commission or omission that could have harmed the patient but did not cause harm as a result of chance, prevention or mitigation . Near miAs a surrogate for WSS, analysis of near miss events may allow organizations to examine the effectiveness of complex systems designed to prevent WSS, without such an event ever occurring . A systeNo Go function of the StartBox System was triggered in 17 of 487 (3%) registered procedures. Quantifying near misses is an important step in understanding the risk of WSS and facilitating a longitudinal review of modified systems and protocols to prevent errors. These errors could have been mitigated by standard prevention protocols, but likely not tracked or reported. Near miss analysis is rare , requiring different room setup or approach to the spine after a midline incision. The StartBox System allowed for further precision to be communicated to the OR team by the surgeon, which was an improvement over previous processes. The system color-coding especially supported the awareness of procedure laterality (including room setup and approach) and contributed to the prevention of this frequent type of WSS.The number of patients in the study is small relative to the volume of surgical procedures at a given institution. The small number of patients limit an effective comparison between sites as well as perform any correlation analyses. Future studies with larger number of patients should provide better opportunity to do so. Additionally, the study at each site was conducted over a relatively short period of time, limiting the ability of the institution to perform in-depth analysis of the near misses and implement considerable systematic change or evaluate effectiveness of changes. Continued evaluation with an increasing number of patients over a longer period of time should help further the appreciation for the incidence of near misses and validate the StartBox System as a robust safety system for WSS prevention.StartBox is designed to prevent wrong site surgery and capture near misses through a real-time, data-driven approach. The system is designed to complement safety checklists, standardize and streamline workflows, integrate computerization, and provide a final constraint to prevent WSS.This evaluation included 487 surgical procedures during which StartBox successfully performed all tasks without technical errors and identified 17 near miss events that could have led to the occurrence of a wrong site surgery. Zero wrong site surgeries occurred in this study.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the US government. DG is an employee of the US government. This work was prepared as part of their official duties and as such there is no copyright to be transferred.DG, BH, and DGS performed procedures that are its subject and wrote sections of the manuscript. All authors contributed conception and design of the research, contributed to manuscript revision, read, and approved the submitted version.BH, DGS, and JD have equity investment in StartBox, LLC. DS is a consultant for StartBox, LLC. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Phaeodactylum tricornutum ICE-H was sequenced using Illumina NovaSeq PE150. The circular mtDNA was 77055 bp in size and encodes 60 genes, contains 24 tRNA genes, 34 protein-coding genes, and 2 rRNA genes. The composition of A + T in ICE-H mtDNA was 65.34%. The phylogenetic relationship of 17 species of plant mitochondria were analyzed using the maximum likelihood method by the MEGA-X. Phaeodactylum tricornutum ICE-H was most closely related to Phaeodactylum tricornutum.In this study, the complete mitochondrial genome (mtDNA) of the Antarctic  Phaeodactylum tricornutum ICE-H belongs to Phaeodactylaceae, a eukaryotic alga that can grow in extreme Antarctic environment. Therefore, there is an urgent need to understand the genome information of ICE-H. Here, we first assemble and annotate the complete mitochondrial genome (mtDNA) of the Antarctic ICE-H.Antarctic sea ice microalgae have crucial effects in maintaining the stability of polar ecosystems . The sample (Accession no. FIO2008697701) is stored in the Key Laboratory of First Institute of Oceanography, Ministry of Natural Resources. We isolated the complete mtDNA of ICE-H and sequenced it based on the NovaSeq PE150 at the Beijing Novogene Bioinformatics Technology Co., Ltd. The mtDNA was assembled by SOAP denovo (version 2.04) , including 34 protein-coding genes, 24 tRNA genes, and 2 rRNA genes. The mtDNA of ICE-H composition is 33.40% for A, 16.71% for C, 17.95% for G, and 31.94% for T. The percentage of A + T in ICE-H mtDNA was 65.34%. The 34 PCGS include COX2-3, NAD1-7, NAD4l, NAD11-a, NAD11-b, RPL6, RPL2, RPL5, RPL14, RPL16, RPS2-4, RPS8, RPS10, RPS12-13, RPS19, ATP6, ATP8, ATP9, COB, TATC. Among the 34 PCGS, except NAD7, COB all protein initiation codons are ATG. The termination codon of most protein-coding genes was TAA (5 of 34 genes) or TAG (3 of 34 genes). The 24 tRNA-coding genes ranged in size from 72 bp to 93 bp.Phaeodactylum tricornutum (HQ840789.1). And the distance between Oryza Sativa Indica Group and Zea mays subsp. was the farthest (Phylogenetic relationship of 17 complete mtDNAs was analyzed using the maximum likelihood method with 1000 bootstraps (Kumar et al. farthest . The res