In healthy CNS, A2A receptors are expressed mainly within the neurons of the basal ganglia. Alterations in A2A receptor function and expression have been noted in movement disorders, and in Parkinson's disease pharmacological A2A receptor antagonism leads to diminished motor symptoms. Although A2A receptors are expressed only at a low level in the healthy CNS outside striatum, pathological challenge or inflammation has been shown to lead to upregulation of A2A receptors in extrastriatal CNS tissue, and this has been successfully quantitated using in vivo positron emission tomography (PET) imaging and A2A receptor-binding radioligands. Several radioligands for PET imaging of A2A receptors have been developed in recent years, and A2A receptor-targeting PET imaging may thus provide a potential additional tool to evaluate various aspects of neuroinflammation in vivo. This review article provides a brief overview of A2A receptors in healthy brain and in a selection of most important neurological diseases and describes the recent advances in A2A receptor-targeting PET imaging studies.Adenosine receptors are G-protein coupled P1 purinergic receptors that are broadly expressed in the peripheral immune system, vasculature, and the central nervous system (CNS). Within the immune system, adenosine 2A (A The disd humans , 24. Howkephalin . A2A recptically  but are ptically  and can 6/2 mice . The abil cortex , 30, hipl cortex , 22, 31,l cortex –38 has ltagonism , 40. TheSCH58261  but this outflow . Similar outflow . Future 2A receptor TMSX ([7-N-methyl-11C]-(E)-8--1,3,7-trimethylxanthine) binding in the putamen was shown to be higher in PD patients with dyskinesias (disease duration: 11.1 ± 7.2 years) compared to healthy controls [11C]TMSX binding compared to healthy controls. [11C]TMSX DVR was, however, increased in the putamen in a follow-up scan after approximately a year of induction of antiparkinsonian therapy compared to the baseline scans, despite the absence of clinical dyskinesias SCH442416, and PET imaging, a significant increase was found in the binding potential in the putamen and the nucleus caudatus in PD patients with levodopa-induced dyskinesias (disease duration: 13.2 ± 5.6) compared to PD patients with levodopa treatment without dyskinesias (disease duration: 6.2 ± 3.4) and to healthy controls [PET imaging using Acontrols . On the kinesias . Similarcontrols .2A receptors in early stages of HD [2A receptors is reported in transgenic mouse models of HD [2A receptor expression in cell cultures by regulating transcription of the A2A receptor gene [2A receptor gene (ADORA2A) rs 5751876 genotype was shown to affect the age of onset of HD in humans [2A receptors rescues cognitive performance impaired by the disease [2A receptor agonists on the other hand have shown to reverse motor deficits [ In vivo A2A receptor-targeting PET imaging using [11C]KF18446 has been used to demonstrate reduced A2A receptor expression in an animal model of HD TMSX have demonstrated that A2A receptor expression is increased in the NAWM of patients with SPMS compared to age- and sex-matched controls  associates with decreased susceptibility risk of MS . Im. Im2A re and PET . The ideironment , 65, 102ironment , 104. Mosk of MS  and withsk of MS , also su2A receptor antagonists such as caffeine or SCH58261 has been shown to significantly reduce clinical scores in multiple mice and rat models of EAE β-CIT-SPECT (single-photon emission computed tomography) can be used for imaging dopamine transporter availability for differential diagnostics of early or atypical PD, [11C]PIB for identifying amyloid pathology in early AD if routine morphological imaging is normal, and [18F]FDG (2-deoxy-2-[fluorine-18]fluoro-D-glucose) for detecting hypometabolism and differentiating dementia with Lewy bodies (DLB) or frontotemporal lobe degeneration (FTD) from AD. In addition, [123I]β-CIT-SPECT may aid in differentiating between DLB and AD. For imaging neuroinflammation, [18F]FDG could theoretically be used for detecting hypermetabolism, but due to its unspecificity, it is of limited value in clinical practice compared to routine MRI imaging and cerebrospinal fluid (CSF) analyses. Thus, when imaging the detailed mechanisms of A2A receptors in neuroinflammation, more specific probes, such as A2A receptor-binding radioligands, are needed.PET imaging of A in vivo PET studies demonstrate greatest A2A receptor ligand binding in the basal ganglia, whereas low radiotracer accumulation was shown in cortical areas and cerebellum TMSX, [11C]Preladenant, [11C]SCH442416, [18F]MNI-444, and [11C]KW6002, have been tested in human subjects. Their chemical structures are presented in In the healthy CNS, humanrebellum , 129–131 binding . Regardied above , 16, 13311C]TMSX is a methylxanthine analog of KF17387. It is the most widely used A2A receptor radioligand and its binding to A2A receptors in humans has been described in the brain [11C]TMSX (previously named KF18446) was first developed by Ishiwata et al. [2A selectivity over A1 and high nonspecific binding [11C]TMSX shows relatively low affinity for the A2A receptor  by approximately 5% when compared with metabolite corrected plasma input [11C]TMSX has been developed and validated [11C]TMSX photoisomerization.Acquiring metabolite corrected plasma input function via arterial cannulation and repeated arterial sampling for the measurement of the radioligand activity and metabolism is considered the golden standard in brain PET image analyses especially with novel ligands without a priori knowledge of the ligand's kinetics and metabolism. However, this methodology can be unpleasant for the study subjects, may be prone to errors, and requires more expert personnel. Therefore, optional methods for obtaining plasma input function have been developed, including independent component analysis  and intema input . Consequalidated . Dosing 11C]SCH442416 (5-amino-7-(3-(4-[11C]methoxy)phenylpropyl)-2-(2-furyl)pyrazolo-1,2,4-triazolopyrimidine) was the first suitable nonxanthine radioligand for the imaging of A2A receptors. In a blocking study with vipadenant (an A2A receptor antagonist), the highest radioligand binding measured as metabolite corrected VT was seen in putamen (VT ~ 0.6 ml/cm3), followed by caudate, nucleus accumbens, thalamus, and cerebellum (VT ~ 0.3 ml/cm3) [2A receptor blocking with vipadenant resulted in notable 3-4-fold reduction in total [11C]SCH442416 binding (VT) in striatal ROIs and also in about up to 2-fold reduction in cerebellum. Two later studies have shown very different specific binding potentials in the putamen when using the cerebellum as a reference region for the estimation of specific radioligand binding. Grachev et al. Preladenant has high affinity for the A2A receptor and >1000-fold selectivity to the A2A over the other adenosine receptor subtypes [11C]Preladenant was recently published [11C]Preladenant has a DVR of 7.9 ± 2.3 in the putamen and shows lower binding in the frontal cortex, thalamus, and cerebellum [11C]Preladenant reduced striatal binding to extrastriatal levels but also reduced extrastriatal binding MNI-444 is the only [18F]-labeled A2A radioligand used in humans. It has relatively high affinity (Ki = 2.8 nM) for the human recombinant A2A receptor KW6002 shows high binding in the striatum, but binding is also detected in the cerebellum and thalamus. In addition, preblocking with A2A receptor antagonist KW6002 reduced [11C]KW6002 binding to A2A receptors in all studied brain regions KW6002 binding was found TMSX binding occurs in putamen and caudate, there appears to be some specific, albeit lower, A2A receptor binding in extrastriatal tissues such as cortical gray matter and cerebellum. The rate of specific binding, calculated as BP/VT, has been reported to be as high as 53% in cerebellum and 37.8–42.7% in cerebral cortex for [11C]TMSX TMSX based on the same algorithm used for [11C]PK11195 studies (SuperPK software) [Even though the highest specific [11C]TMSX . Similaroftware) . Importaoftware) .2A receptor antagonists and agonists in a variety of neurological conditions. A2A receptors are ubiquitously expressed in various areas of the CNS, but their significance in the context of the different CNS diseases still needs clarification. Pathological processes in CNS diseases are particularly difficult to investigate for reasons such as the difficulties in obtaining representative biopsies from the brain. PET imaging, on the other hand, provides an excellent opportunity to evaluate disease-specific pathology in vivo, by allowing quantitative study of the receptors of interest in an appropriate pathological environment in situ. With the increasing variety of A2A receptor-binding PET ligands available for use in human in vivo PET imaging, there is good likelihood that PET imaging will improve our understanding of the involvement of A2A receptors in the pathophysiology and pathogenesis of brain diseases, both in the neuronal compartment of the basal ganglia and in relation to inflammation, such as in progressive MS. Groups of patients can be studied cross sectionally at various stages of a given disease or, alternatively, PET imaging can be applied longitudinally to evaluate alterations in the A2A receptor in the course of the disease or in response to treatment. PET imaging of neuroinflammation has relied heavily on TSPO-binding radioligands, but methodological challenges related to TSPO-imaging has directed the field to actively seek alternative imaging probes. A2A receptor PET imaging provides one such alternative that is worth further exploring.There is increasing interest in the therapeutic development of A


Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway.The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the  Legionella pneumophila uses such a strategy to exploit ubiquitylation, a conserved post-translational modification that is mediated by E3 ubiquitin ligases. L. pneumophila encodes molecular mimics of host E3 ligases, including the effector protein RavN, thereby subverting the ubiquitylation pathway for its own benefit during infection. Using protein crystallography, we show that the fold of RavN has only residual resemblance to conventional eukaryotic E3s, yet its mode of interaction with E2 enzymes, host proteins that are important for the ubiquitin transfer reaction, has been preserved throughout evolution. Inspired by the discovery of RavN, we performed an in silico fold homology search and discovered several additional E3 ligase candidates within the effector repertoire of L. pneumophila that, until now, had remained hidden due to lack of primary sequence similarity. Our study supports the hypothesis that E3 ligases are a vital part of the virulence program of L. pneumophila, and that these effectors, despite having undergone extensive evolutionary changes, have retained features that are critical for their biological function, including the ability to hijack host factors that are part of the ubiquitylation machinery.Bacterial pathogens often hijack conserved host pathways by encoding proteins that are molecular mimics of eukaryotic enzymes, thus tricking the host cell into surrendering its resources to the bacteria. Here, we show that the intracellular pathogen  Ubiquitylation is one of the most significant and versatile post-translational modifications in cell biology. It shapes almost all cellular processes including protein homeostasis and trafficking, signal transduction, and cell cycle progression –3. The hHomologous to the E6-AP Carboxyl Terminus)-type E3s form a transient intermediate with ubiquitin via a Cys residue in their active site prior to ubiquitin transfer onto the substrate protein, while RING  or U-box-type E3s function as a scaffold by bringing the E2-ubiquitin conjugate into close proximity with the target protein in order for Ub transfer to occur, notably without the formation of a covalent E3-Ub intermediate. Given the importance of ubiquitylation in eukaryotic cell physiology and protein regulation, it is not surprising that this post-translational modification is exploited by a variety of pathogens that encode molecular mimics of E3 ligases .–48.1-123E3 and L2E3) in a cross-brace arrangement , 4947, 4 species . Closer  species  are clusnterface .I8S, RavNL43S, and RavNP47S) in which each interface residue was individually replaced by serine, as well as a double mutant (RavNL43S/P47S) and a triple mutant (RavNI8S/L43S/P47S), and examined their E3 ligase activity in the in vitro reconstitution assay  or two residues (FLAG-RavNL43S/P47S) within the putative E2 binding site was sufficient to severely reduce the poly-ubiquitylation activity of the proteins. Substituting all three residues nearly abolished the poly-ubiquitylation levels of FLAG-RavNI8S/L43S/P47S -tagged ubiquitin in transiently transfected HEK293T cells, and ubiquitylated species were detected using anti-HA antibody after anti-FLAG immuno-precipitation . While F43S/P47S . This rel lysate  was primS. cerevisiae INVSc1 on media plates containing galactose, substitution of individual interface residues  of RavN resulted in a partial recovery of yeast growth. S. cerevisiae producing RavN variants with substitutions in either two (RavNL43S/P47S) or three (RavNI8S/L43S/P47S) interface residues grew at levels comparable to cells producing no RavN (vector control). Thus, inhibition of yeast growth upon RavN overproduction was most likely a result of its E3 ligase activity. Taken together, our cell-based and in vitro assays confirmed that even though the three-dimensional structure of RavN has been significantly altered during evolution compared to related U-box E3 ligases, the E2-E3 interface has remained conserved and plays a critical role for RavN’s function.Overproduction of RavN did not cause any detectable cytotoxicity in mammalian cells , but str1-140, but not RavN1-100, functioned as E3 ligase  was fully active. Thus, the flexible α3 helix is unlikely to be directly involved in the E3 ligase activity of RavN.Our domain mapping revealed that RavN3 ligase , suggestric unit . The angor α3B∠C . This suactivity . The onl1-110 resulted in a drastic reduction in E3 ligase activity  to a degree similar to that of the I8S substitution in RavNI8S/L43S/P47S (lane 5). When introduced into wild-type RavN, mutation F103A caused a slight yet noticeable decrease in E3 ligase activity of RavNF103A , thus expanding the E3 repertoire of this organism to ten. This unexpected abundance of a variety of structurally and functionally diverse ubiquitin ligases suggests that harnessing the host ubiquitylation machinery is of greater importance for the virulence program of L. pneumophila than was initially thought. Prior to our study, six L. pneumophila proteins had been experimentally confirmed to exploit host ubiquitylation. Four  had been discovered based on primary sequence homology to known E3s  that encode a single FLAG affinity tag, followed by restriction-mediated cloning via the HindIII and XhoI sites into the vector pcDNATM5/FRO/TO (Invitrogen). pYES2-ravN was generated by subcloning ravN into the yeast expression vector pYES2/NTA (Invitrogen) digested with BamHI and XbaI. The pcDNA6.2-FLAG vector was generated by replacing the emgfp sequence in pcDNA6.2-EmGFP (Invitrogen) with the sequence encoding a single FLAG affinity tag using the EcoRV and PspXI site. Genes encoding L. pneumophila E3 ligase candidates were cloned into pcDNA6.2-FLAG via Gateway Cloning. Genes encoding RavN or effector proteins with single or multiple amino acid substitutions were generated by QuikChange site-directed mutagenesis (Agilent Technologies) using the respective primers  were cloned into the pGST-Parallel2 vector . After mechanical lysis of bacterial cell using a cell breaker (Microfluidics M-110P), His6-tagged effector proteins were enriched on TALON Metal Affinity Resin (Clontech), and non-specifically bound proteins were removed by washing with Wash buffer . Human embryonic kidney (HEK293T) cells (ATCC) were suspended in Wash buffer containing protease inhibitor, and lysed with 40–50 strokes in a dounce homogenizer. The post-nuclear supernatant was collected by centrifugation at 15,000 rcf for 10 min at 4°C. The pulldown assay was performed by incubating the TALON affinity resin containing 50 μg effector protein overnight at 4°C on a rotator with post-nuclear supernatant of HEK293T cells containing 5 mg of total protein. The resin was washed five times with Wash buffer with 0.05% Tween-20 and mixed with 2X SDS sample buffer. Eluted proteins were separated by SDS-PAGE and detected by silver staining (Bio-Rad) or on an immunoblot using anti-ubiquitin antibody.The pDEST17 constructs were introduced into 6- or GST-tagged RavN or His6-tagged Lpg2452 were overproduced in the presence of 0.2 mM IPTG for 16 hours at 20°C and purified with TALON Metal Affinity Resin (Clontech) or Glutathione Sepharose 4B  following the manufacturer’s protocol. Proteins were dialyzed against 1X PBS with 1 mM β-mercaptoethanol. In vitro ubiquitylation was performed using the Ubiquitinylation kit (Enzo Life Sciences). In brief, a 10 μL reaction mixture containing 1X ubiquitinylation buffer, 5 mM Mg-ATP, 1 mM DTT, 2.5 μM biotinylated ubiquitin, and 100 nM E1 was combined with 1 μM to 2.5 μM of the various E2s and 1 μM purified E3 (RavN or Lpg2452). The reaction was incubated at 37°C for one hour or the indicated time, mixed with an equal volume of 2X SDS sample buffer, and incubated at 94°C for 10 min. The proteins were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and ubiquitylated products were detected with HRP-conjugated streptavidin.His5 HEK293T cells were transiently transfected with pcDNA5/FRO/TO-FLAG-ravN or pcDNA6.2-FLAG constructs encoding FLAG-tagged effector proteins with pRK5-HA-ubiquitin. After overnight incubation, the cells were treated with or without 10 μM MG132 for six hours and were harvested by scraping. Cells were lysed in lysis buffer  at 4°C for 30 min, and the FLAG-tagged effector proteins were enriched on ANTI-FLAG M2 affinity gel (Sigma) according to the manufacturer’s protocol. The resin was mixed with SDS sample buffer, proteins were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with anti-ubiquitin (Abcam or Life Technologies), anti-ubiquitin Lys48 linkage-specific antibody (Abcam), anti-HA (Abcam), or anti-FLAG (Sigma) antibody.2x10E. coli BL21(DE3) containing the pGST-Parallel2 vectors  and lysed by high-pressure homogenization at 27 Kpsi (Constant System Ltd). The cell lysate was subjected to centrifugation at 50,000 x g for 45 min, and the supernatant was loaded on a gravity column with glutathione-sepharose beads . The beads were washed with PBS-BME and subsequently with buffer A . Proteins were eluted from the beads after overnight incubation with tobacco etch virus (TEV) protease (in buffer A) which cleaves off the N-terminal GST-tag. Proteins of interest were diluted to a final concentration of 50 mM NaCl and passed over a 5 mL HitrapQ column  equilibrated in 50 mM Tris-HCl pH 8.0, 50 mM NaCl and 10 mM BME, and eluted using a 50–1,000 mM NaCl gradient. Finally, the protein was purified on a Superdex 200 16/60 size-exclusion column  in 50 mM Tris-HCl pH 7.4, 150 mM NaCl and 10 mM BME. Peak fractions were pooled, concentrated with an Amicon Centrifugal filter (Millipore), flash-frozen in liquid nitrogen, and stored at -80°C.RavN and RavNProteolysis-resistant fragments of RavN were identified by incubating full-length RavN with trypsin at a ratio 250:1 for 90 minutes at 37°C. The resulting products were isolated by gel filtration on a Superdex 200 16/60 column. Two main peaks were observed and analyzed by mass spectrometry. Molecular weight determination was performed by MALDI-TOF analysis  by using sinapinic acid for sample preparation, and spectra were acquired in linear mode. The proteolytically resistant fragments had a molecular mass of 12,830 Da and 10,896 Da, which corresponded to the residues 1 to ~109 of RavN  from the TEV cleavage site) and the C-terminal region spanning amino acids ~118 to 212, respectively.1-123 were obtained by hanging-drop vapor diffusion method at 18°C with a reservoir solution containing 1.5 M Ammonium sulfate, 0.1 M NaCitrate pH 5.4 and 0.2 M K/Na Tartrate. Crystallization drops contained 1 μL protein sample at 70 mg/mL, mixed with 1 μL reservoir solution. The crystals were cryoprotected by brief soaking in mother liquor containing 20–25% (vol/vol) glycerol and flash cooled in liquid nitrogen. For structure solution, crystals were incubated with the cryoprotectant solution containing NaI 400 mM for 4 minutes. Diffraction data of native and NaI derivative crystals were collected at 100 K either at the Diamond Light Source  beamline I04 or at the ALBA beamline XALOC  [Crystals of RavN, Spain) .http://www.pymol.org/).Data sets were integrated and scaled using XDS . The cryS. cerevisiae strain INVSc1 cells were transformed with the resulting constructs using the lithium acetate/PEG transformation method described in the manufacturer’s protocol (Life Technologies). Transformants were grown at 30°C to saturation in synthetic liquid media containing 2% (w/v) glucose without uracil (-Ura). Cultures were normalized to an OD600 = 1, and 10-fold serial dilutions were spotted on synthetic media plates (-Ura) containing glucose (repressing) or galactose (inducing), and the growth was monitored after 48 hours of incubation.Genes encoding RavN or RavN variants were expressed from pYES2/NTA (Invitrogen), a vector that contains a galactose-inducible (GAL1) promoter. L. pneumophila effector proteins were analyzed on the HHpred server using default parameters (https://toolkit.tuebingen.mpg.de/#/tools/hhpred) [The amino acid sequences of 296 /hhpred) . Effectohttp://www.rcsb.org/pdb) under ID code 5MIY.Coordinates and structure factors have been deposited in the Protein Data Bank . (B) Growth-phase-dependent production of RavN in either Lp02 or Lp02ΔravN by immunoblot using anti-RavN antibody (GenScript). LidA served as loading control [L. pneumophila strains Lp02, Lp03 (T4SS-), and Lp02ΔravN for 2 hours, and bacterial colony-forming units were determined in a plating assay after 2, 24, 48, and 72 hours. Results are an average from two independent experiments. (D) RavN shows cytosolic distribution pattern. GFP-tagged RavN was produced in transiently transfected COS-1 cells, and its localization was analyzed by fluorescence microscopy. GFP was used as control. (E) RavN is enriched in the cytosolic fraction in infected macrophages. U937 cells challenged with Lp02 at an MOI of 100 for two hours were subjected to membrane fractionation. Calnexin and GAPDH served as marker proteins for the membrane (M) or cytosolic (C) fraction, respectively. P: post-nuclear supernatant.(A) Phylogenetic tree of the genus ted from  showing  control . (C) Rav(TIF)Click here for additional data file.S2 FigLegionella species was performed with Clustal Omega [1-123, as revealed by its crystal structure ((A) Sequence alignment of RavN homologs from ten different al Omega . Secondatructure , are ind(TIF)Click here for additional data file.S3 Fig1-123. The MALS-based molecular weight profile for the elution peak is shown (blue solid line), along with the predicted MW of the monomer  as reference. (B) Final 2Fo−Fc electron density calculated after the final refinement run (contoured at the 2σ level) (blue), with the final RavN model overlaid. (C) The anomalous difference electron-density map contoured at 5.0 σ showing the signals of the Iodine scattering atoms.(A) SEC-MALS analysis of RavN(TIF)Click here for additional data file.S4 FigThe E2 binding interface of RavN and CHIP (circled) are similar with respect to their topology and electrostatic potential, with positively (blue) and negatively (red) charged residues highlighted. The displayed electrostatic potential was calculated using APBS .(TIF)Click here for additional data file.S5 FigI8S/L43S/P47S, and RavNL43S/P47S/F103A. The spectra were plotted with Molar Ellipticity θ (in deg x cm2 x dmol-1) against wavelength (in nm). (B) In vitro ubiquitylation assay using untagged RavN, RavNI8S/L43S/P47S, and RavNL43S/P47S/F103A shown in (A). UbcH5a was added as E2 enzyme. Poly-ubiquitylation was detected by HRP-conjugated streptavidin (left), and total amounts of RavN present in each reaction were detected using RavN-specific antibody (right). (C) Ubiquitylated species in FLAG-RavN in (A) Circular dichroism spectra of wild-type RavN, RavN(TIF)Click here for additional data file.S6 FigAll alignments were based on HHpred results. The top row indicates the predicted secondary structure of the query effector protein, and the bottom row displays the secondary structure of the template protein from the Protein Data Bank . (A) Alignment of Lpg2370 with the RING-type E3 ligase FANCL (PDB ID 4CCG). (B) Alignment of Lpg2577 with the Bre1 RING finger domain (PDB ID 4R7E). The cysteine residues that form the zinc finger are highlighted in yellow. (C) Alignment of Lpg2498 with the HECT-type E3 ligase E6AP (PDB ID 1C4Z). (D) Alignment of the N-terminal 120 amino acid residues of Lpg2452 with SidC (PDB ID 4OOJ). The cysteine residue that forms the catalytic Cys-His-Asp triad is highlighted in yellow.(TIF)Click here for additional data file.S7 FigThe immunoblot shown in (TIF)Click here for additional data file.S8 FigShown here is a proposed model for E2-Ub-RavN interaction and the dynamic motion of the flexible α3 helix that connects the E3 ligase domain of RavN (gray) with the putative target-binding C-terminal domain (yellow).(TIF)Click here for additional data file.S9 Figlpg2452, sidC, and sdcA in different Legionella genomes. Note the apparent lack of synteny, except in the genome of L. pneumophila where lpg2452 and sidC/sdcA coexist.Overview of the presence (+) or absence (-) of (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Methods(PDF)Click here for additional data file.


Prevalence of susceptibility epitopes was higher in patients and was not exclusively a result of linkage disequilibrium. Residues DRB1 Q70, DQB1 L26 and A57 and a 10 amino acid epitope of DQA1 were the most significant in discriminating risk alleles. An extended haplotype containing these epitopes was carried by 92% of our patient cohort. Sharing of susceptibility epitopes could also explain the absence of risk haplotypes in patients. Finally, many significantly associated epitopes were non-pocket residues suggesting that critical immune functions may exist spanning further from the binding pockets.The contribution of specific HLA Class II alleles in type 1 diabetes is determined by polymorphic amino acid epitopes that direct antigen binding therefore, along with conventional allele frequency analysis, epitope analysis can provide important insights into disease susceptibility. We analyzed the highly heterogeneous Cypriot population for the HLA class II loci of T1DM patients and controls and we report for the first time their allele frequencies. Within our patient cohort we identified a subgroup that did not carry the DRB1*03:01-DQA1*05:01-DQB1*02:01 and DRB1*04:xx-DQA1*03:01-DQB1*03:02 risk haplotypes but a novel recombinant one, DRB1*04:XX-DQA1*03:01-DQB1*02:01 designated DR4-DQ2.3. Through epitope analysis we identified established susceptibility (DQB1 A Type 1 diabetes (T1D) is an organ-specific autoimmune disease affecting the insulin producing β cells of the pancreas, leading to absolute insulin deficiency  with a gAlthough close to 60 diabetes-associated genetic linkages have been reported to date , numerouHLA molecules function to present antigenic peptides to T cells and thus have a central role in immune cell activation and autoimmune disease. The peptide-binding grooves of HLA molecules are made of amino acid residues arranged in pockets; these amino acids are highly polymorphic and, either as single or groups of continuous or non-continuous residues, create millions of possible epitopes. These epitopes determine the repertoire of peptides a given HLA allele can present. Class I molecules  have binding grooves made of six pockets. Class II molecules  are heterodimers comprised of α and β chains creating binding grooves with four major pockets. In the HLA-DR heterodimer polymorphism is found only on the β chains whereas both α and β chains of the HLA-DQ and HLA-DP heterodimers are polymorphic. In addition, not all pairings between α and β chains are allowed explaining structural correlates and associations or non-associations with disease ,22.In addition to conventional allele frequency studies, the systematic analysis of HLA epitopes has recently been highlighted as a critical component in providing a better understanding of genetic susceptibility to T1D . Indeed,26 and A57 but also susceptibility residue DRB1 Q70 and alternative protective R70 not previously reported to be associated with T1D. Finally, we show that an extended risk haplotype of HLA class II susceptibility epitopes identified in this study DRβQ70-DQβL26A57DQαY11R52R55F61T64I66L69V/L76 H129E/K175 could account for 92% of our patient cohort.In the current study we investigated the HLA frequencies in a cohort of Cypriot T1D patients, a population that has not been studied to date. We propose that studying genetically heterogeneous populations such as the Cypriot population can be critically informative in allowing further validation of established risk alleles and epitopes or uncovering new ones. We report here, for the first time, the allele frequencies in Cypriot T1DM patients and a new HLA Class II risk haplotype DR4-DQ2.3. Furthermore, we identified previously reported HLA class II susceptibility residues including DQB1 L-12, OR = 6.56) and *04:05 , HLA-DQB1*02:01  and 03:02 (pcorr. = 1.19x10-12 and OR = 6.58), HLA-DQA1*03:01  and HLA-DPB1*03:01 . In contrast, the alleles most commonly found in the control population, and therefore deemed protective, were HLA-DRB1*14:01 , *11:04 , *10:01  and *16:02 , HLA-DQB1*03:01  and *05:03 , HLA-DQA1*01:01  and HLA-DPB1*04:02 .We used a dataset of 170 T1D patients and 192 control subjects for whom high-resolution HLA genotyping was performed at 4 classical major histocompatibility complex class II loci DRB1, DQA1, DQB1 and DPB1. Allele frequency analysis  Tables sThe high-resolution HLA genotyping dataset of patients and control subjects was imported into the SKDM HLA Tool for analysis to identify epitopes associated with T1D. Both pocket and non-pocket residues were investigated for each allele and a number of them were found significantly associated. The susceptibility epitopes with the lowest p values (p ≤ 0.001) and highest OR (OR ≥ 1.5) and the resistance epitopes with the lowest p values (p ≤ 0.001) and lowest OR (OR ≤ 1.5) are summarized in 57 . This is found in HLA-DQB1*02:01 and *03:02 alleles which were significantly more common in patients according to the allele frequency analysis (57) was identified as the residue most strongly associated with resistance . Two of the HLA-DQB1 alleles  sharing this residue were significantly more frequent in controls. Similarly, the presence of leucine (L) in position 26 was the second strongest residue associated with susceptibility , whilst tyrosine (Y)  or glycine (G)  at the same position confer resistance. L26 is found in HLA-DQB1*02:01 and *03:02 alleles which are more common in patients. HLA-DQB1 susceptibility residues F47  and R70  were also found in the same position as resistance residues Y47  and G70 . In contrast, resistance residues A13 , Y37 , T28  were not in the same position as susceptibility ones.The strongest association was shown for the previously reported HLA-DQB1 residue Aanalysis . Asparti70 had the strongest association . This residue is shared between most high risk alleles including DRB1*03:01, *04:05 and *04:01, which were all significantly more common in patients  and K71  were also significantly associated with susceptibility. HLA-DRB1 H13 is shared by a number of HLA-DRB1 *04:xx alleles that confer high risk. HLA-DRB1 *04:01 which shares all three of these epitopes had the highest OR 10.07. HLA-DRB1 resistance residues R70  or D70  and R71  are at the same positions as susceptibility counterparts. Resistance residue R70 in particular had the lowest OR and is shared among two protective alleles HLA-DRB1*10:01 and *14:01 which were significantly less common in patients. The presence of arginine (R) at position 74 is associated with susceptibility , whereas glutamic acid (E) at the same position is significantly associated with resistance . Finally, residues R30 and A38 were also significantly associated with resistance and had the next lowest OR after R70 .The DRB1 locus contained the next most significant HLA residue associated with susceptibility to T1D; HLA-DRB1 Qpatients . In addi11, R55, I66 and L69  and the previously reported R56 and V76  most frequently observed in patients  are at the same positions as susceptibility counterparts and are shared between HLA-DQA1* 01:03 and *01:01 alleles, which were significantly more frequent in controls.A number of DQA1 residues were strongly associated with T1D; Y, OR 6.8 . All arepatients . Further57 had the strongest association with T1D  . A gene dose effect was apparent since inheritance of one copy of A57 decreased the probability of disease as shown by the lower OR , in the same way inheritance of one copy of the susceptibility residue D57  was not as protective as inheritance of two copies but still lowered probability of disease compared to inheritance of one copy of A57. However, resistance appears to be dominant since inheritance of only one copy of D57 still conferred a lower probability of diabetes (OR 0.08). A similar effect was observed for DQB1 L26, inheritance of two copies is strongly associated with T1D , inheritance of one copy greatly decreased the probability of disease  and two copies of G26 is protective . Inheritance of two copies of the HLA-DRB1 Q70 susceptibility residue was also strongly associated with T1D  and probability decreased with inheritance of one copy . Among the two alternative resistance residues identified for this position, inheritance of R70 appears to be more protective than inheritance of D70; one copy of R70 conferred lower probability of disease  than one  or two copies of D70 . The probability of disease in the case of inheritance of two copies of R70 was not calculated as this genotype was not present in the population.The effect of homozygosity or heterozygosity of class II residues identified to be associated with susceptibility or resistance was analyzed. Homozygosity of susceptibility epitope DQB1 AR 100.6) , whilst 70 and the DQβ A57 was compared between patients and control subjects (70 epitope (2.9%) or only the DQβ A57 epitope (4.1%) or none of the two epitopes (2.9%). Among control subjects the highest percentage (38.5%) had none of these susceptibility epitopes . These findings suggest that our observations are not only due to linkage disequilibrium (LD) between the two loci. In addition, it was shown that these DR/DQ susceptibility epitopes were not in LD in patients (p = 0.001) but were in LD in controls (p = 1.8x10-6).The prevalence of having both the highest susceptibility residues DRβ Qsubjects . The maj26, R70, L85, E86, T89, but more importantly the most significant susceptibility residue A57. Hence the presence of either HLA-DQB1*02:01 or HLA-DQB1*03:02 makes the patient homozygous for the A57 residue, associated with the highest OR in the zygosity analysis (p = 1.25x10-24 OR 100.6).Having identified a number of susceptibility and resistance residues associated with T1D we sought to further dissect our patient cohort with regards to their HLA genotypes aiming to explain disease susceptibility by the presence of shared susceptibility residues. More specifically, among our T1D patients the majority (135 of 170 or 79%) carried at least one or both of the susceptibility alleles DR3-DQ2.5 and DR4-DQ8 . HoweverCarrying at least one copy of any of the risk haplotypes DR3-DQ2.5, DR4-DQ8 or DR4-DQ2.3 could account for 92% (157 of 170) of patients . HoweverTo investigate whether epitopes differentiate associated alleles, allele sequences were retrieved and aligned using the IMGT/HLA database of the European Bioinformatics Institute. Within these sequences we noted all the susceptibility and resistance residues, both pocket and non-pocket, identified in this study and also found to have a proposed function according to literature –29. The 9, V11, H13, Y26, N37 and R74 that are associated with diabetes are amino acids that are part of binding pockets , whereas protective HLA-DRB1*03:01 contains aspartic acid (D) in the same position. In addition, risk associated HLA-DQB1*03:02 also contains the susceptibility epitope L26. Finally, a sequence of 10 amino acid residues could differentiate risk and protective HLA-DQA1 alleles. Epitope Y11R52R55F61T64I66L69V/L76H129E/K175 was exclusive to risk associated alleles and was not observed in any protective alleles.Overall, all risk associated HLA alleles contain more susceptibility residues and protective alleles contain more protective residues. Some residues, however, might not be as critical as others. For example, HLA-DRB1 susceptibility residue E70-DQβL26A57DQαY11R52R55F61T64I66L69V/L76 H129E/K175 epitope haplotype compared with only 27% of the control subjects. A very small percentage of patients had only the DRβ Q70 residue (2.9%) or only the DQβ A57 residue (4.1%) or none of the two (2.9%). Among control subjects the highest percentage (73%) had none of these susceptibility epitopes compared to only 8% of patients .We counted the number of individuals carrying an extended haplotype containing all the HLA class II risk associated epitopes in our patient-control cohort . The epith and 91st in Cypriots. We thus believe that the Cypriot population represents an excellent study sample that can allow further dissection of T1D disease susceptibility. Using our highly diverse cohort of Cypriot patients, we were able to identify a new recombinant predisposing haplotype, DR4 –DQ2.3, carried by 13% of our patients, but only 4.7% of control subjects. This haplotype failed to reach significance in a large T1D Genetics Consortium investigating HLA-DR-DQ haplotypes in 607 Caucasian families and 38 Asian families /[(b + ½)(c + ½)], and the significance of its derivation from unity is estimated by Fisher’s exact test. HLA epitopes with a corrected P value ≤ 0.001 were defined as statistically significant. In addition, odds ratio (OR) ≥ 1.5 determined susceptibility while OR ≤ 0.5 determined resistance.S1 TableThe HLA-DRB1 typing of the patient and control populations. The table includes presence in the population and frequency, allele number and frequency, delta difference between the T1D and CTL population frequencies, a corrected P-value and the Odds Ratio (OR).(DOCX)Click here for additional data file.S2 TableThe HLA-DQB1 typing of the patient and control populations. The table includes presence in the population and frequency, allele number and frequency, delta difference between the T1D and CTL population frequencies, a corrected P-value and the Odds Ratio (OR).(DOCX)Click here for additional data file.S3 TableThe HLA-DQA1 typing of the patient and control populations. The table includes presence in the population and frequency, allele number and frequency, delta difference between the T1D and CTL population frequencies, a corrected P-value and the Odds Ratio (OR).(DOCX)Click here for additional data file.S4 TableThe HLA-DPB1 typing of the patient and control populations. The table includes presence in the population and frequency, allele number and frequency, delta difference between the T1D and CTL population frequencies, a corrected P-value and the Odds Ratio (OR).(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.S10 Table(DOCX)Click here for additional data file.S11 Table(DOCX)Click here for additional data file.S12 Table(DOCX)Click here for additional data file.S13 Table(DOCX)Click here for additional data file.


During the 2013–14 influenza season in the United States, influenza activityDuring September 29, 2013–May 17, 2014, World Health Organization and National Respiratory and Enteric Virus Surveillance System collaborating laboratories in the United States tested 308,741 specimens for influenza viruses; 53,470 (17.3%) were positive . Of the During the 2013–14 season, pH1N1 viruses were the predominant viruses in circulation nationally, with fewer influenza B viruses and influenza A (H3) viruses also identified. Using the percentage of specimens testing positive for influenza to determine the peak of influenza activity, the peak occurred during surveillance week 52  nationally; however, differences among the 10 U.S. Department of Health and Human Services regionsWhereas pH1N1 activity peaked between late December and late January, influenza B activity occurred later in the influenza season. Influenza A viruses predominated until late March, and influenza B viruses became the most commonly identified virus nationally during the week ending March 29, 2014 (week 13). The intensity and timing of influenza B activity varied geographically. One region (Region 4) never reported a single week during which influenza B viruses predominated, whereas influenza B viruses were predominant in Region 6 from week 9  through week 20 . During the late season increase in influenza B activity, the overall number of influenza A viruses decreased; however, the proportion of influenza A viruses subtyped as (H3) increased. In week 13  influenza A (H3) viruses became the predominant influenza A virus nationally. Region 2 was most heavily impacted with late season influenza B activity, whereas Region 1 in the northeastern United States reported most late season influenza A (H3) activity. Region 2 identified 21.4% of all influenza B viruses nationally; Region 1 identified 21.4% of all influenza A viruses subtyped as (H3) nationally.During the 2013–14 influenza season, one case of human infection with an H3N2v virus occurred during week 40  in a child from Iowa with known direct exposure to swine. The child fully recovered, and no additional cases were identified in family members or other close contacts.CDC antigenically characterized 2,905 influenza viruses collected and submitted by U.S. laboratories since October 1, 2013, including 2,036 pH1N1 viruses, 426 influenza A (H3N2) viruses, and 443 influenza B viruses. Of the 2,036 pH1N1 viruses tested, 2,033 (99.9%) were antigenically similar to A/California/7/2009, the influenza A (H1N1) component of the 2013–14 Northern Hemisphere influenza vaccines. Three viruses (0.1%) of the 2,036 tested showed reduced titers with ferret antiserum raised against A/California/7/2009. Of the 426 influenza A (H3N2) viruses tested, 406 (95.3%) were antigenically similar to A/Texas/50/2012, the influenza A (H3N2) component of the 2013–14 Northern Hemisphere vaccines. Twenty (4.7%) of the 426 tested showed reduced titers with antiserum produced against A/Texas/50/2012.Of the 443 influenza B viruses tested, 323 (72.9%) belonged to the B/Yamagata lineage, and 322 (99.7%) were antigenically similar to B/Massachusetts/2/2012, the influenza B component of the 2013–14 Northern Hemisphere trivalent and quadrivalent influenza vaccines. One (0.3%) virus showed reduced titers with antiserum produced against B/Massachusetts/2/2012. The remaining 120 (27.1%) influenza B viruses belonged to the B/Victoria lineage and were antigenically similar to B/Brisbane/60/2008, the influenza B component of the 2013–14 Northern Hemisphere quadrivalent influenza vaccine.Since October 1, 2013, a total of 6,294 influenza virus specimens have been tested for resistance to influenza antiviral medications. All 508 influenza B viruses and 683 influenza A (H3N2) viruses tested were sensitive to both oseltamivir and zanamivir. Among the 5,103 pH1N1 viruses tested for resistance to oseltamivir, 59 (1.2%) were resistant, and all of the 1,890 viruses tested for resistance to zanamivir, including the 59 oseltamivir-resistant viruses, were sensitive. Resistance to the adamantanes (amantadine and rimantadine) persisted among influenza A viruses currently circulating globally (the adamantanes are not effective against influenza B viruses).The Food and Drug Administration’s Vaccines and Related Biological Products Advisory Committee has determined that the 2014–15 influenza vaccines used in the United States have the same antigenic composition as those used in 2013–14. The trivalent vaccines should contain an A/California/7/2009-like (2009 H1N1) virus, an A/Texas/50/2012-like (H3N2) virus, and a B/Massachusetts/2/2012-like (B/Yamagata lineage) virus. The committee also recommended that quadrivalent vaccines contain a B/Brisbane/60/2008-like (B/Victoria lineage) virus pdm09–predominant season since the emergence of the virus in 2009, and also had later-season influenza B activity. The highest hospitalization rates were among adults aged ≥65 years, which is consistent with previous influenza seasons; hospitalization rates among those aged 50 to 64 years were significantly higher than in all years since the 2009 pandemic. Nearly all of the influenza virus specimens sent to CDC for antigenic characterization were similar to the components of the 2013–14 Northern Hemisphere influenza vaccine. The Food and Drug Administration has recommended that the 2014–15 influenza vaccines used in the United States have the same antigenic composition as those used in 2013–14.What are the implications for public health practice?Influenza surveillance, including for novel influenza viruses, should continue throughout the summer months, and health-care providers should consider influenza as a cause of respiratory illness even outside the typical season. Although influenza viruses typically circulate at low levels during the summer months, timely empiric antiviral treatment is recommended for patients with severe, complicated, or progressive influenza illness and those at higher risk for influenza complications; treatment can be considered for others if it can be started within 48 hours of illness onset.ILINet data are used to produce a weekly jurisdiction-level measure of ILI activity,State and territorial epidemiologists determine the geographic distribution of influenza in their jurisdictions using all available data sources through a weekly influenza activity code.CDC monitors hospitalizations associated with laboratory-confirmed influenza virus infections using the Influenza Hospitalization Surveillance Network (FluSurv-NET).As of May 30, 2014, among the FluSurv-NET adult patients for whom medical chart data were available, 89.0% had at least one underlying medical condition. The most frequent underlying medical conditions identified were obesity (42.9%), metabolic disorders (36.0%), and cardiovascular disease (34.6%). Among children hospitalized with laboratory-confirmed influenza and for whom medical chart data were available, 57.0% had at least one underlying medical condition. The most commonly identified conditions were asthma (25.4%) and neurologic disorders (14.1%). Among the 882 hospitalized women of childbearing age (15–44 years), 197 (22.3%) were pregnant.During the 2013–14 influenza season, the percentage of deaths attributed to pneumonia and influenza (P&I) exceeded the epidemic thresholdFor the 2013–14 influenza season, 96 laboratory-confirmed, influenza-associated pediatric deaths were reported from 30 states, New York City, and Chicago. The deaths included 18 children aged <6 months, 24 aged 6–23 months, eight aged 2–4 years, 27 aged 5–11 years, and 19 aged 12–17 years; mean and median ages were 6.0 years and 4.6 years, respectively. Among the 96 deaths, 79 deaths were associated with influenza A virus infections , 13 deaths were associated with influenza B viruses, two deaths were associated with an influenza virus for which the type was not determined, and two deaths were associated with an influenza A and influenza B virus coinfection. Of 90 children with known medical history, 49 (54.4%) had at least one high-risk medical condition. Neurologic disorders (29 [32.2%]) and pulmonary disease (17 [18.9%]) were the most commonly identified conditions.Since influenza-associated pediatric mortality became a nationally notifiable condition in 2004, the total number of influenza-associated pediatric deaths has ranged from 35 to 171 per season; this excludes the 2009 pandemic, when 348 pediatric deaths were reported to CDC during April 15, 2009–October 2, 2010.The 2013–14 influenza season peaked in late December with pH1N1 viruses predominating nationally and in all 10 regions. Activity decreased through January and February, but a late season increase in influenza B activity occurred in March, and influenza B viruses became the predominant virus nationally in week 13 . Nearly all of the influenza virus specimens sent to CDC for further antigenic characterization were similar to the components of the 2013–14 Northern Hemisphere vaccines.After several recent influenza A H3N2)–predominant seasons, 2013–14 was the first pH1N1–predominant season since the 2009 pH1N1 pandemic. During the 2009 pandemic, adults aged 50–64 years had the highest mortality rate and second highest influenza-associated hospitalization rate, and during the 2013–14 season, adults again were at high risk of severe influenza illness. The cumulative incidence of hospitalization among adults aged 50–64 years during the 2013–14 season was well above the range of rates seen in seasons following the pandemic, whereas hospitalization rates in all other age groups were within the range seen in recent years. This age distribution of hospitalizations is likely attributable to several factors, including lack of cross-protective immunity to pH1N1 and lower influenza vaccination coverage among persons in this age group (–predominTesting for seasonal influenza viruses and monitoring for novel influenza A virus infections should continue year-round, as should specimen submission to CDC for further antigenic and genetic analysis and antiviral resistance monitoring. Human infections with novel influenza A viruses were identified in greater numbers during the summer months of 2012 and 2013  and mighAlthough influenza activity in summer in the United States typically is low, cases of influenza, and even influenza outbreaks, are detected in the United States throughout the summer. Health-care providers should remain vigilant and consider influenza as a potential cause of summer respiratory illnesses, and also consider treatment with influenza antiviral medications for those at high risk for influenza-associated complications, as recommended by the Advisory Committee on Immunization Practices . Health-http://www.cdc.gov/flu/weekly. Additional information regarding influenza viruses, influenza surveillance, influenza vaccine, influenza antiviral medications, and novel influenza A virus infections in humans is available at http://www.cdc.gov/flu.Influenza surveillance reports for the United States are posted online at CDC weekly and are available at


Initiation of efavirenz-based combination antiretroviral therapy (cART) is associated with Vitamin D deficiency, but the risk factors including efavirenz pharmacokinetics for cART-induced severe vitamin D deficiency (SVDD) and the impact of anti-tuberculosis (TB) cotreatment are not explored. We investigated the prevalence of SVDD in HIV and TB-HIV coinfected patients and associated risk factors for treatment-induced SVDD.th, 16th, and 48th week of cART. Plasma efavirenz concentrations were determined at 4th and 16th weeks of cART.Treatment-naïve Ethiopian HIV patients with (n = 102) or without (n = 89) TB co-infection were enrolled prospectively and received efavirenz-based cART. In TB-HIV coinfected patients, rifampicin-based anti-TB treatment was initiated 4 or 8 weeks before starting cART. Plasma 25-hydroxyvitamin D (25 [OH]D), cholesterol and 4-beta hydroxycholesterol concentrations were measured at baseline, 43 levels than HIV-only patients at baseline. TB co-infection, low Karnofsky score, high viral load, and high CYP3A activity as measured by plasma 4β-hydroxycholesterol/cholesterol ratios were significant predictors of low 25 (OH)D3 levels at baseline. In HIV-only patients, initiation of efavirenz-based cART increased the prevalence of SVVD from 27% at baseline to 76%, 79%, and 43% at 4th, 16th, and 48th weeks of cART, respectively. The median 25 (OH)D3 levels declined from baseline by −40%, −50%, and −14% at 4th, 16th, and 48th weeks of cART, respectively.TB-HIV patients had significantly lower plasma 25 (OH)D3 levels, but the initiation of efavirenz-based cART increased the prevalence of SVDD from 57% at baseline to 70% and 72% at the 4th and 16th weeks of cART, respectively. Median plasma 25 (OH)D3 declined from baseline by −17% and −21% at week 4 and 16 of cART, respectively.In TB-HIV patients, previous anti-TB therapy had no influence on 25 (OH)D3 level at baseline is associated with TB co-infection and HIV diseases progression. Initiation of efavirenz-based cART is associated with high incidence of SVDD, whereas rifampicin based anti-TB therapy co-treatment has no significant effect. Supplementary vitamin D during cART initiation may be beneficial for HIV patients regardless of TB coinfection.Our results indicate low plasma cholesterol, high CYP3A activity, and high plasma efavirenz concentrations as significant predictors of early efavirenz-based cART-induced vitamin D deficiency. Low plasma 25 (OH)D Vitamin D3 is formed naturally from 7-dehydrocholesterol, a cholesterol precursor, in the skin upon exposure to sunlight. Thus factors influencing 7-dehydrocholesterol levels may result in altered vitamin D and cholesterol levels. Efavirenz-based combination antiretroviral therapy (cART) is associated with changes in both vitamin D–9 and cholesterol concentrations, but it is unclear whether there is a correlation between the variation in vitamin D and cholesterol levels during cART and whether this effect is related to drug concentrations.Vitamin D deficiency is associated with many chronic illnesses, including autoimmune, cardiovascular, and infectious diseases like tuberculosis (TB), HIV disease progression, and mortality.–13 Efavirenz and rifampicin, the cornerstones of first-line cART and anti- TB treatments, respectively, are potent inducers CYP2B6 and CYP3A enzymes and transporter proteins.,14,15 Efavirenz is mainly metabolized by genetically polymorphic CYP2B6 and to some extent by CYP3A enzymes. Vitamin D is involved in the regulation of CYP3A and CYP2B6 enzyme expression.,17 However, CYP3A catalyzes the 4-hydroxylation of 25 (OH)D3, and hence CYP3A induction may contribute to drug-induced vitamin D deficiency.,19 Long-term CYP2B6 and CYP3A induction by efavirenz is influenced by pharmacogenetic factors relevant for efavirenz disposition.,15,20–22 Accordingly, host genetic factors affecting the plasma concentration of CYP3A inducers may also potentially affect the vitamin D levels in patients on long-term treatment with efavirenz or rifampicin. Indeed, a CYP2B6 genotype-based efavirenz dose adjustment was recommended recently to optimize treatment outcomes.,24 Implication of inter-individual variations in efavirenz plasma concentration for efavirenz-induced vitamin D deficiency is yet to be investigated.TB is the most common opportunistic infection and the leading cause of death in people living with HIV. Concomitant HIV and TB treatments are challenging because significant drug–drug interactions, overlapping toxicities, and immune reconstitution inflammatory syndrome.–9 Poor absorption, lower exposure to the sunshine, and darker skin pigmentation are known risk factors for low 25 (OH)D levels. Data regarding predictors of 25 (OH)D levels during cART are scarce in HIV patients living in tropical areas, where there is an all-year round sunshine. The risk factors for cART-induced severe vitamin D deficiency and the implication of anti-TB co-treatment remain to be investigated, especially in sub-Saharan Africa, where both HIV and TB are major public health problems. In the present study, we hypothesized that high CYP3A induction by rifampicin co-treatment and inter-individual variability in efavirenz disposition may play a role for between-patient variability in vitamin D status during cART. Therefore, the objectives of this study were to identify the prevalence and associated risk factors for low 25 (OH)D levels and severe vitamin D deficiency before starting treatment and during efavirenz-based cART alone or together with rifampicin-based anti-TB co-therapy in HIV or TB-HIV co-infected patients, respectively.Previous studies reported that efavirenz-based cART lowers the 25 (OH)D level.22.1The study design was prospective, comparative, observational, open-label, parallel assignment, 2-arm pharmacogenetic and pharmacokinetic cohort study to identify the prevalence and risk factors for severe vitamin D deficiency at baseline and during efavirenz-based cART with or without rifampicin-based anti-TB therapy. The study was conducted between June 2008 and June 2011 at HIV and TB clinics in Addis Ababa (latitude 9– 1’ N), Ethiopia.All patients gave written, informed consent to participate in this study. The study protocol received ethics approvals from the Institutional Review Board (IRB) of School of Medicine, Addis Ababa University, National Ethics Review Committee of Ethiopia. The study also received approval from IRB of Karolinska Institutet  and was conducted as per International Conference for Harmonization-Good Clinical Practice (ICH-GCP) guidelines.2.2 Briefly, for the main study, newly diagnosed HIV-infected (n = 285) and TB-HIV co-infected (n = 208) patients were recruited prospectively and enrolled in parallel and followed up to 48 weeks. The eligibility criteria were ≥18 years of age, not pregnant, and CD4 count ≤200 cells/mm3. None of the study participants received isoniazid prophylaxis or other TB treatment for 2 years before enrollment. Treatment adherence was assessed by self-report.This study was conducted as one of the substudies designed under the umbrella of the broad clinical research project entitled “The HIV-TB Pharmagene Study” in Ethiopia. Details of the main study design, patient enrolment process, and inclusion criteria with follow-up and drug treatments were reported previously.th and 16th weeks of cART, were used to monitor the change in vitamin D during cART. The sample size was calculated for each treatment group considering a moderate effect size, E = 0.3, to detect the change in vitamin D levels before and after efavirenz exposure (paired t test) with 80% of study power and α = 5%, the desired sample size was calculated as 87 in each group. Plasma sample collected at 48 weeks of cART from 42 HIV-only patients was also available and used.For the present study, a total of 191 patients (102 TB-HIV co-infected patients and 89 HIV only infected patients), with complete set of plasma samples collected at baseline, and at the 42.3All HIV patients received cART (600 mg efavirenz-based cART containing either zidovudine/lamivudine/efavirenz or stavudine/lamivudine/efavirenz). Plasma samples for the determination of plasma cholesterol, 4β-hydroxycholesterol, and 25 (OH)D were taken before the initiation of treatment (week 0) and at weeks 4, 16, and 48 of treatment.All TB-HIV patients n = 102) initiated rifampicin-based anti-TB treatment 4 (n = 69) or 8 weeks (n = 33) before the initiation of efavirenz-based cART. The short course anti-TB treatment consisted of an initiation phase with rifampicin/isoniazid/pyrazinamide /ethambutol for 2 months followed by a continuation phase with rifampicin/isoniazid for 4 months. Samples for the determination of plasma cholesterol, 4β-hydroxycholesterolm and 25 (OH)D concentrations were collected before starting anti-TB treatment (corresponding to week -4 or -8 weeks of cART), at the initiation of cART (week 0) and weeks 4 and 16 of cART. The study population, follow-up period, and study sampling time points are presented in Figure 02 initia3 <20 ng/mL (50 nmol/L), and levels between 21 and 29 ng/mL (51–72.5 nmol/L), considered as insufficient. Severe vitamin D deficiency (SVDD) was defined as plasma 25 (OH)D3 level <25 nmol/L. The predicting variables were patient's clinical and laboratory parameters at baseline and during treatment, plasma cholesterol and efvairenz concentrations, and CYP3A enzyme activity as defined by 4β-hydroxylcholesterol/cholesterol ratio.Outcome variables were plasma 25 (OH)D level and vitamin D status; vitamin D deficiency (VDD) defined as circulating levels of 25 (OH)D2.43 and 25 (OH)D2 in plasma were determined by isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously. Briefly, 25 (OH)D was extracted from plasma using a Hybrid SPE precipitation 96-well plate  and isopropanol in acetonitrile . 25 (OH) D3/D2 Plasma Control (level 1) and 25 (OH)D3/D2 Plasma Calibration Standard were used as quality control and calibrator, respectively . 2H6–25-hydroxyvitamin D3 was used as internal standard . Analysis was done on Acquity Quattro Premier LC-MS/MS  system equipped with a UPLC BEHC18 column . The mobile phase was a gradient of 50% to 95% acetonitrile in water (0.1% formic acid). The lower limit of detection (LOD) of 25 (OH)D3 was 2.5 nmol/L and the lower limit of quantification (LOQ) 7 nmol/L. The linear range was 2.5 to 625 nmol/L. The relative standard deviation (CV) was 16% (at 33.5 ± 5.4 [n = 39] [mean ± sd]). The LOQ of 25 (OH)D2 was 12 nmol/L.25 (OH)D2.5Cholesterol was determined in plasma using a commercial enzymatic method  on a Roche/Hitachi Modular Instrument. The between-day variation was 1.3% at 5 mmol/L.2.6,26,29 Briefly, protein precipitation was performed using acetonitrile containing internal standards . Analysis was performed using a Synergi Fusion RP chromatography column  and mobile phases containing ammonium acetate , methanol, and acetonitrile. The lower limits of quantification in plasma were 10.0 ng/mL. The efavirenz calibration range was 10 to 10000 ng/mL. The method was validated according to the FDA validation guidelines and fulfilled all criteria concerning accuracy, precision, recovery, linearity, and stability.Plasma samples for determination of efavirenz were collected at weeks 4 and 16 of cART in both treatment groups. Plasma efavirenz concentrations were determined using LC-MS/MS as described previously.2.73 levels between treatment groups was done using the Mann–Whitney test. Plasma 25 (OH)D3, cholesterol, efavirenz concentrations, and 4β-hydroxycholesterol/cholesterol ratios were log-transformed (base 10) before applying the t test, repeated measure analysis of variance (ANOVA), and regression analysis. Pairwise comparison of data from baseline within and between treatment groups was made using paired and unpaired t test, respectively. For each patient, the percent change in plasma 25 (OH)D3 level from baseline to the 4th, 16th and 48th weeks of cART was calculated using the following formula: Comparison of median plasma 25 (OH)D3 levels over time. Univariate followed by multivariate linear regression analysis was performed to identify predictors of low plasma 25 (OH)D3 levels at baseline and during treatment. Predictor variables that resulted in a P value <0.1 in the univariate regression analysis was entered into a backward stepwise multivariate regression analysis to identify significant predictors in the final model. Likewise logistic regression was done to determine predictors of severe vitamin D deficiency before and during cART. Statistical analyses were performed using Statistica version 12  and SPSS Statistics  software, version 23.0. GraphPad Prism version 5.0 for Windows  was used for graphical presentations. A P value <0.05 was considered significant.Repeated measure ANOVA was used to analyze the change in log plasma 25 (OH)D33 + 25 (OH)D2 in plasma. Very few patients had 25 (OH)D2 levels above the lower LOQ; hence, all statistical calculations were done using only plasma 25 (OH)D3 data.The baseline sociodemographic, clinical, and laboratory characteristics of study participants stratified by treatment group are presented in Table 3.13 levels at baseline and during cART and the median percent change in plasma 25 (OH)D3 levels during therapy between the 2 treatment groups are presented in Table 3 concentration was significantly lower in TB-HIV coinfected patients than in HIV patients without TB. In the HIV-only cohort, initiation of efavirenz-based cART dramatically lowered the median plasma 25 (OH)D3 levels by −40%, −50%, −14% at week 4, 16, and 48 of cART, respectively D3 level and the median percent change of 25 (OH)D3 from baseline by 4th and 16th of weeks of cART was −17% and −21%, respectively.Comparisons of the median plasma 25 (OH)D3 levels from baseline after 4 weeks of efavirenz-based cART alone versus 4 or 8 weeks of rifampicin-based anti-TB therapy alone are presented in Figure 3 levels compared to HIV-only patients at baseline . Initiation of cART in TB-HIV patients on anti-TB therapy lowered the mean plasma 25 (OH)D3 levels, and there was no significant difference in the mean plasma 25 (OH)D3 levels between the 2 treatment groups at weeks 4 and 16 of cART.Comparisons of change in log plasma 25 (OH)D3 levels profile from baseline during efavirenz-based cART alone versus rifampicin-based anti-TB co-treatment is presented in Figure 3 (P < 0.001). Paired t tests indicated significantly lower plasma 25 (OH)D3 levels at week 4  and at week 16  compared with the baseline pretreatment value. As cART continued, the plasma 25 (OH)D3 levels were restored, and no significant difference from baseline was found after 48 weeks of cART (P = 0.12) in HIV-only patients.The overall change in the mean log plasma 25 (OH)D3 (P < 0.0001). Paired t tests indicated significantly lower 25 (OH)D3 level at week 4  and week 16  compared to the baseline value. Data on plasma 25 (OH)D3 at week 48 of cART in TB-HIV co-infected patients were not available.In TB-HIV co-infected patients, within-treatment group analysis indicated that efavirenz-based cART significantly reduced mean log plasma levels of 25 (OH)D3.23 levels at the study enrollment. Despite receiving cART and anti-TB therapy, none of the patients achieved a sufficient (>72.5 nmol/L) plasma 25 (OH)D3 level during the study follow-up period.All patients had deficient (92%) or insufficient (8%) plasma 25 (OH)Dth, 16th, and 48th weeks of efavirenz-based cART in each treatment group is presented in Table P < 0.001). In the HIV-only cohort, initiation of efavirenz-based cART significantly increased the proportion of patients with SVDD from 27% at baseline to 76% (95% CI = 67.1%–84.5%) at week 4, and 79% (95% CI = 70.3%–87.3%) at week 16, and 42.5% (95% CI = 32.2%–52.7%) at week 48 cART. Whereas in TB-HIV patients on rifampicin-based anti-TB therapy, initiation of efavirenz-based cART co-treatment increased the proportion of patients with SVDD from 57% at baseline to 70% (95% CI = 69.7%–78.5%) at week 4 and 72% (95% CI = 63.6%–80.9%) at week 16 of cART.The prevalence of SVDD before starting treatment (at baseline) and at the 43.33 level particularly in HIV-only patients treated with efavirenz-based cART . The median and IQR of efavirenz plasma concentrations in HIV-only and TB-HIV patients at week 16 of cART were 1328 (980–1947) ng/mL and 1236 (826–2013) ng/mL, respectively (P = 0.26).The median (IQR) of efavirenz plasma concentrations in HIV-only and TB-HIV patients at week 4 of cART was 1200 (841–1846) ng/mL and 1574 (984–2041) ng/mL, respectively , and high serum alkaline phosphatase. In a stepwise multivariate regression analysis, low Karnofsky score, low albumin level, high viral load, and high 4β-hydroxycholesterol to cholesterol ratio remained significant predictors of low 25 (OH)D3 concentration at baseline.Linear, regression analysis was used to identify predictors of low plasma 25 (OH)Dne Table . In a unP value <0.1 in the univariate analysis D level soon after efavirenz-based cART initiation D3 level at baseline with TB-co-infection and HIV disease progression; efavirenz-based cART significantly reduces plasma 25 (OH)D3 levels and this effect is more pronounced when given alone than with rifampicin-based anti-TB therapy; low plasma cholesterol level, high CYP3A activity, and high plasma efavirenz concentration are predictors of early cART-induced low 25 (OH)D3 level.We performed a prospective observational study to identify predictors of a low 25 (OH)D,31 However, lack of sunshine-derived vitamin D deficiency is not expected in countries around the equator.,33 Ethiopia is located in east Africa close to the equator (3 degree N to 14.8 degree latitude) and there is abundant sunshine to form vitamin D all year round. This study was conducted in Addis Ababa, the capital city of Ethiopia, located at latitude 9 degree N. In the present study, we found no significant influence of seasonal variation (rainy versus dry seasons) on plasma 25 (OH)D3 concentrations. Our finding is consistent with previous findings from countries located near the equator.,33Seasonal variation affects vitamin D status, particularly in countries far from the equator, where the lower angle of the sun and more cloud cover during the winter result in less UV-B exposure and hence low production of vitamin D in skin.,36 HIV patients and patients with active TB have lower levels of 25 (OH)D3 than healthy controls and patients with latent TB.,38 Accordingly, all patients who participated in this study had deficient (92%) or insufficient (8%) plasma 25 (OH)D3 levels at study enrollment, and none of them achieved a sufficient plasma 25 (OH)D3 level (>72.5 nmol/L) during a 1-year cART follow-up period. The prevalence of vitamin D deficiency in our treatment-naive HIV-only and TB-HIV patients from Ethiopia is quite high compared to other reports from HIV patients in East Africa. Low prevalence of vitamin D insufficiency at baseline in TB-HIV co-infected (41%) and HIV (35%)-only infected patients from Uganda has been reported. Likewise, only 9.2% and 43.6% of HIV patients from Tanzania were vitamin D-deficient and -insufficient, respectively. The finding of a high prevalence of SVDD in Ethiopian HIV patients might be because our study population consisted of very ill patients with low CD4 cell count (<200 cells/mm3) at baseline and a majority presented HIV stage 3 or 4 indicating progression to AIDS  and insufficiency (49%) in Ethiopian healthy adolescents and children was reported previously.3 and SVDD at baseline was significantly associated with high viral load, which is a marker for HIV disease progression. Our finding is in line with previous reports from the literature describing association of vitamin D deficiency with HIV disease progression. Variations in the dietary source containing vitamin D may also contribute to between-population differences in vitamin D status. TB co-infected HIV patients had significantly lower levels of 25 (OH)D3 than patients without TB co-infection. Our finding further indicates that severe vitamin D deficiency in HIV patients is a risk factor for the development of active TB.,43In our regression analysis Table , low pla3 levels from baseline. However, a significant reduction of plasma 25 (OH)D3 levels was noted soon after initiating cART in both HIV-only and TB-HIV co-infected patients treated. The effect of efavirenz-based cART in lowering 25 (OH)D3 levels was more pronounced when given alone than when given together with rifampicin-based anti-TB therapy. In HIV only cohort, initiation of efavirenz-based cART increased the proportion of patients with SVDD by 3-fold at week 4 of cART, which persisted until week 16. As therapy continued, vitamin D levels were gradually restored, but 42% of the patients still had SVDD after 48 weeks of cART (Table Treatment with rifampicin-based anti-TB treatment (up to 8 weeks) before the start of cART in TB-HIV co-infected patients resulted in no significant change in 25 (OH)D3 level may be related to an increased catabolism of 25 (OH)D through CYP3A4 enzyme induction. In line with this, we found a significant correlation between CYP3A enzyme activity, as measured by 4β-hydroxycholesterol to cholesterol ratio, and low 25 (OH)D concentration both at baseline and week 4 of cART. But as therapy continued, no significant association between CYP3A activity and vitamin D status was observed. We previously reported a significant association between plasma efavirenz concentration and 4β-hydroxycholesterol/cholesterol ratio in HIV patients. In the present study, we found a significant correlation between plasma efavirenz concentration and 25 (OH)D3 concentration. Vitamin D is involved in the regulation of CYP3A enzyme expression.,19 CYP3A4 enzyme activity and expression are induced by 1,25-(OH)D3 via the vitamin D receptor. In addition, CYP3A catalyzes the 4-hydroxylation of 25 (OH)D3 and hence CYP3A induction may contribute to drug-induced vitamin D deficiency.,19Previous in vitro studies suggested that a low 25 (OH)D Thus, it is plausible to expect a more pronounced effect of rifampicin in lowering the 25 (OH)D3 level compared to efavirenz-based cART alone. However, we found no significant effect of rifampicin-based anti-TB therapy (given before the initiation of cART), on the vitamin D status D3 alongside the corresponding plasma cholesterol level in each patient before starting therapy and at different time points during treatment. Notably, TB-HIV co-infected patients had significantly lower pretreatment plasma concentration of both cholesterol and vitamin D compared to HIV-only patients. A similar pattern of change in plasma cholesterol level and vitamin D status over time was observed during cART, particularly when given alone D3 concentration by early initiation of efavirenz might be secondary to change in cholesterol concentration. In line with this, a recent in vitro study reported that treatment of cells with cholesterol resulted in a 3-fold increase in vitamin D relative to cholesterol synthesis, demonstrating that cholesterol feeds back via 7-dehydrocholesterol reductase (DHCR7) increasing vitamin D production. Indeed our results indicate that rifampicin-based anti-TB therapy alone significantly increased the plasma cholesterol level D3 and cholesterol levels restored gradually, which may be because of overall improved health status. In diverse HIV-infected populations, Vitamin D insufficiency and deficiency are associated with HIV disease progression, and virological failure after antiretroviral therapy initiation. Recent randomized clinical trials indicated that vitamin D supplementation improved CD4 recovery and vitamin D repletion suggesting potential benefit on immunologic recovery during cART.,49Increased plasma levels of 25 (OH)D4.1This study has some limitations. First, the study participants consisted of patients who had CD4 cell count <200 at baseline, following the WHO and Ethiopian national HIV treatment guideline valid during the study period. Thus, the result may not be directly extrapolatable to patients with high CD4 cell count at baseline. Second, since vitamin D status is influenced by genetic variations, darker skin, exposure to the sunshine, and dietary sources, the study result from a single study location and Ethiopian population may not be applicable for genetically diverse non-black populations living in the nontropical zone.53 at baseline. Early initiation of efavirenz-based cART is associated with low plasma 25 (OH)D3 levels and high incidence of SVDD, which sustains up to 16th week of cART. This effect is most pronounced when given alone than with rifampicin-based anti-TB co-treatment. As cART continues, 25 (OH)D3 levels restore gradually with time. Rifampicin-based anti-TB treatment initiated before cART has no significant effect on the 25 (OH)D3 levels. High plasma efavirenz concentration, high CYP3A activity, and low plasma cholesterol are predictors of early cART-induced low plasma 25 (OH)D3 level. Considering the very low levels of 25 (OH)D3 in both patient groups, supplementary vitamin D may be beneficial not only for TB-HIV patients, but also for HIV-only patients at the initiation of cART and anti-TB treatment.The finding of this study suggests TB incidence and HIV disease progression are associated with low levels of 25 (OH)D


Here; we report that the American cockroach (Periplaneta americana) expresses a second type 1 tyramine receptor (PeaTAR1B) in addition to PeaTAR1A . When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP]i. Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine.The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP] Periplaneta americana, is an established model organism in neurobiological, physiological, and toxicological studies i. With an EC50 of 6.3 nM, activation of PeaTAR1B was much more sensitive to tyramine than the PeaTAR1A receptor (EC50 = 350 nM) which was recently characterized using the same heterologous expression system i. Whether P. americana expresses type 2 tyramine receptors has to be addressed in future studies.In conclusion, the occurrence of two functional type 1 tyramine receptors with different pharmacological profiles in P. americana aminergic receptors i were monitored with the Ca2+-sensitive fluorescent dye Fluo-4. Cells were grown in 96-well dishes to a density of approximately 2 × 104 cells per well and were loaded at room temperature with Fluo-4 AM as described previously ethanesulfonic acid (HEPES), and 10 mM glucose, pH 7.4 (NaOH)) containing 100 µM IBMX. Measurements on cells expressing PeaTAR1B-HA were performed in the presence of 1 µM NKH477 , since PeaTAR1B-HA was predicted to inhibit adenylyl cyclase activity. The plate was transferred into a fluorescence reader  to monitor Fluo-4 fluorescence. The excitation wavelength was 485 nm. Fluorescence emission was detected at 520 nm. Concentration series of various biogenic amines and synthetic receptor ligands were added once Fluo-4 fluorescence had reached a stable value in each well. Receptor ligands  were all purchased from Sigma . The changes in Fluo-4 fluorescence were recorded automatically. Concentration–response curves were established from at least three independent experiments with quadruplicate or octuplicate measurements. Data were analyzed and displayed using Prism 5.04 software .A cell line expressing the highest amount of the receptor was used to examine PeaTAR1B receptor activity by Calar Ca2+ ,99,113. eviously ,73,99. AP. americana contains two genes which both code for type 1 tyramine receptors, PeaTAR1A [50 6.3 nM) compared to PeaTAR1A (EC50 350 nM). Pharmacologically PeaTAR1B was efficiently blocked by mianserin and yohimbine, whereas other potential receptor antagonists were non-effective. Whether PeaTAR1A and PeaTAR1B represent the complete inventory of tyramine receptors in this insect remains to be established.The cockroach PeaTAR1A  and PeaT


Prostate cancer (PC) is the most common cancer in men. Elevated levels of E3 ligase, E6-Associated Protein (E6AP) were previously linked to PC, consistent with increased protein expression in a subset of PC patients. In cancers, irregular E3 ligase activity drives proteasomal degradation of tumor suppressor proteins. Accordingly, E3 ligase inhibitors define a rational therapy to restore tumor suppression. The relevant tumor suppressors targeted by E6AP in PC are yet to be fully identified. In this study we show that p27, a key cell cycle regulator, is a target of E6AP in PC. Down regulation of E6AP increases p27 expression and enhances its nuclear accumulation in PC. We demonstrate that E6AP regulates p27 expression by inhibiting its transcription in an E2F1-dependent manner. Concomitant knockdown of E6AP and p27 partially restores PC cell growth, supporting the contribution of p27 to the overall effect of E6AP on prostate tumorigenesis. Overall, we unravelled the E6AP-p27 axis as a new promoter of PC, exposing an attractive target for therapy through the restoration of tumor suppression. Prostate cancer (PC) is one of the leading causes of cancer death among men , 2. Whenin vitro and in vivo, and that this effect is mediated, in part, through an E6AP-target we previously identified [E6AP (E6–Associated Protein) is the prototype of the E3 ligases characterised by a C-terminal HECT domain . Howeverentified : the tumentified . HoweverCDKN1B gene and it regulates cell cycle progression from quiescent to G1 and from G1 to S phase by inhibiting cyclin-dependent kinases (CDKs)/cyclin complexes [CDKN1B gene are rare in cancers. This suggests that p27 is largely deregulated in cancer at the expression level and by altered subcellular localization . P27, aomplexes , 15. Botomplexes , 17. Conomplexes . Moreoveomplexes . Interesiewed in ).In this study, we have demonstrated a new link between E6AP and p27 in PC. Our data demonstrates this link in human PC samples, in a xenograft mouse model and in cultured PC cell lines. We identified the levels at which E6AP regulates p27 and propose a mechanistic explanation for its loss in PC. Importantly, our study exposes a novel E6AP-p27 axis contributing to PC pathogenesis and exposes a new therapeutic opportunity.To investigate the role of E6AP-p27 axis in a clinical setting, we analysed E6AP and p27 protein levels by immunohistochemistry in two cohorts of PC patients using tissue micro-arrays (TMAs). The two independent TMAs represent patients from different stages of the disease; TMA1 contains biopsies sampled from 47 PC patients with Gleason score 6-9 and TMA2 contains 117 biopsies from PC patients with Gleason score 3-7. The product of the proportion  of positive cancer cells and their nuclear staining intensity  was then derived to create a histoscore of 0-12 for each sample. The analysis of the two TMA cohorts revealed that patients with high levels of E6AP (mean histoscore ~10 for TMA1 and 7 for TMA2) predominantly express low levels of p27 (mean histoscore 4) Figure  than in in vitro. DU145 and PC-3 PC cells were transduced with a doxycycline (Dox)-inducible conditional GFP-tagged lentiviral construct with a short hairpin against E6AP (shE6AP) or a hairpin Control (sh-Ctrl). Knockdown of E6AP resulted in elevated levels of p27 in DU145  mice results in tumor growth attenuation and extended survival [in vitro and in vivo, suggesting that E6AP negatively regulates p27 protein expression in PC cells.We next evaluated the effect of E6AP knockdown on p27 levels in an survival . Westernin vivo. Analysis of p27 expression by immunohistochemistry in DU145 xenografts collected at ethical endpoint as described in Figure P27 activity is not only regulated by its protein expression levels, but also by its subcellular localization , 17. P27p27 mRNA expression by quantitative real-time PCR (qRT-PCR). Knockdown of E6AP in DU145 and PC-3 cells significantly increased p27 mRNA levels compared with sh-Ctrl cells  or a deletion mutant lacking the E2F1 binding site (p27E2F1-mut) as a control [E2F1-WT promoter by 1.7 fold compared with the sh-Ctrl cells but did not have any effect on cells transfected with p27E2F1-mut promoter  were identified with this profile, whereas only 26% of tumors in TMA2 n=117) showed this profile , while in vitro and in vivo. Countering this, restoration of p27 expression partially rescued the cell growth inhibition induced by E6AP knockdown  were maintained in DMEM , supplemented with 10% fetal bovine serum (Gibco) and 0.1% Penicillin/Streptomycin . Cells were incubated in a humidified incubator at 37°C with 5% CO2. For passaging, cells were washed with PBS-EDTA and trypsinized with 0.25% trypsin (Gibco). Trypsin was neutralized using DMEM with 10% fetal bovine serum. Trypsinized cells were subsequently centrifuged at 1000 rpm for 5 min at 4°C, and the resultant pellet was suspended in media for sub-culturing. Cell counts were performed using a haemocytometer.For the tumor cell line xenograft experiments, the experiments were performed as explained in .Lentivirus expressing shRNA were previously described in . The seqDU145 and PC-3 cells were seeded and treated with Dox for 96hrs (Dox was topped up every 48hrs). Immunoblotting was performed as previously described . AntibodImmunohistochemistry was performed as described in . Antibodt method. All the assays were performed in technical triplicates in at least three independent experiments.Cells were collected and RNA was isolated using TRIzol (Life Technologies) as per the manufacturer's protocol. cDNA was synthesized from the RNA using M-MLV reverse transcription kit . Primers used: human p27 (forward 5′-GGCCTCAGAAGACGTCAAAC-3′ and reverse 5′-ACAGGATGTCCATTCCATGA-3′); human RPL37a  forward (5′-GCCAGCACGC CAAGTACAC-3′ and reverse 5′-CCCCACAGCTC GTCTCTTCA-3′). PCR was performed on StepOnePlus PCR machine  using Fast SYBR Green Master Mix (Applied Biosystems). Gene expression was calculated using the relative CImmunofluorescence assay was performed as explained in . PhotogrE2F1-WT) or a deletion mutant lacking the E2F1 binding site (p27E2F1-mut) as a control (provided by Prof. Jiandong Chen) and Renilla luciferase construct as the internal control. Luciferase assay was performed as described in [PC-3 cells expressing inducible shE6AP or sh-Ctrl were transfected with p27 promoter containing an E2F1 binding site (p27ribed in .ChIP assay was performed as described previously  using an


Background Systemic cancer spread is preceded by the establishment of a permissive microenvironment in the target tissue of metastasis - the premetastatic niche. As crucial players in establishment of the pre-metastatic niche, myeloid derived suppressor cells (MDSC) release S100A8/A9, an exosomal protein that contributes to metastasis, angiogenesis, and immune suppression. We report the application of antibody-based single-photon emission computed tomography (SPECT) for detection of S100A8/A9 in vivo as an imaging marker for pre-metastatic tissue priming.Methods A syngeneic model system for invasive breast cancer with (4T1.2) or without (67NR) the tendency to form lung metastasis was established in BALB/c mice. A SPECT-probe has been generated and tested for visualization of S100A9 release. Tumor-associated changes in numbers and fuction of immune cells in pre-metastatic tissue were evaluated by flow cytometry and confocal microscopy.Results S100A8/A9 imaging reflected MDSC abundance and the establishment of an immunosuppressive environment in pre-metastatic lung tissue . The S100A8/A9 imaging signal in the pre-metastatic lung correlated with the subsequent metastatic tumor burden in the same organ . CCL2 blockade and the consecutive inhibition of premetastatic niche establishment was clearly depicted by S100A9-SPECT .Conclusion We report S100A8/A9 as a potent imaging biomarker for tumor-mediated immune remodeling with potential applications in basic research and clinical oncology. To enable systemic S100A9 imaging and explore the potential of S100A9 as a translatable diagnostic marker, we have now developed S100A9-specific single photon emission computed tomography (SPECT) imaging. Using a murine model of metastatic breast cancer, we present the first in vivo visualization of systemic tumor-mediated effects on immune cells and establish S100A8/A9 as a surrogate marker for immunomodulation in the context of pre-metastatic lung tissue priming. Whole-body S100A8/A9 SPECT imaging mirrored the establishment of a metastasis-permissive microenvironment and allowed for assessment of the immunosuppressive state in pre-metastatic lung tissue.We recently established target-specific As PN establishment also includes endothelial activation, several factors associated with vascular reorganization have been exploited as indicators of premetastatic processes, including VEGFR and avb3 -CD19-CD11b+CD14+monocytes  in 4T1.2- and 67NR-tumor-bearing mice -CD19-CD11b+CD14+)  derived from spleens of 4T1.2-bearing mice secreted a significantly higher amount of S100A8/A9 as compared to cells from 67NR-bearing mice or matched samples of granulocytes (Gr-1+CR2+CD115-)  breast cancer cell lines 4T1.2 and 67NR1x10Mouse organs were dissected at different time points and cell suspensions prepared for flow cytometry analyses and cell sorting. Sorted monocyte populations were plated in 96-well plates and treated with 50ng/ml recombinant mouse CCL2 (Invitrogen). After 48hrs, supernatants were screened for S100A8 using ELISA  according to manufacturers' instructions.For some experiments, tissues snap-frozen and 4-5 μm thick sections were cut and stored at -80°C for further immunofluorescence staining and confocal microscopy analysis.in vivo procedures were covered by project licences, issued by the UK Home Office and conducted according to the Animals Scientific Procedures Welfare Act.For CCL2 blocking, 4T1.2 tumor-bearing mice received an intraperitoneal injection of 100µg goat polyclonal anti-CCL2 antibody  every other day beginning on day 4 after tumor inoculation, following established protocols The protocol for labelling antibodies for SPECT imaging has been described in detail earlier in vivo imaging, the required volume (1mg in 1mL) of the conjugated compound was incubated with 150 MBq In-111 chloride  for 1h at room temperature and subsequently purified from free In-111 and transferred to PBS using a PD-10 desalting column . The purity of the labeled compound was assessed using HPLC. In-111 labeled aS100A9-DTPA was injected in amounts, corresponding to around 10 MBq per animal.For The rabbit-derived control antibody of irrelevant specificity was labeled accordingly.in vivo imaging experiments were conducted on a dedicated small animal SPECT system , calibrated for In-111 emitted β-radiation. Tracers were applied in amounts, corresponding to 10 MBq/animal intravenously into the tail vein 24h before in vivo imaging. The mean scan time was about 60min with an initial morphologic whole body spiral CT and a consecutive whole body SPECT scan with a frame time of 60s.All Animals were held under Isoflurane inhalation anaesthesia (2% Isoflurane in air) for the whole scan time.in vivo imaging, mice were either kept for longitudinal follow up examinations or sacrificed for tissue collection.Data were reconstructed and analysed using in built VivoQuant Software and presented as percentage of the injected dose (%ID) per selected region of interest (ROI). For individual organ analysis, 3D ROIs were drawn on the anatomic CT images. The ROI size was identical for all parallel experiments. Mean values and standard deviation were calculated for %ID. After cγ blocking solution in MACS buffer (Miltenyi) for 15 min. Cells were incubated for 30min with different combinations of fluorochrome-conjugated antibodies to membrane markers and then fixed/permeabilised. Intracellular staining for Foxp3 or S100A8 was carried out for 30min. Stained cells were acquired in a LSR Fortessa  and analysed with FlowJoSoftware . Details of antibodies used are shown in the supplementary information.All incubation steps were carried out at 4°C. Cells were stained with live dead  dye in PBS for 20min, incubated with Fp values of <0.05 were considered statistically significant.All data are presented as the mean ± standard deviation. Non-parametric Mann-Whitney U test or one-way ANOVA with Bonferroni post-test were used for statistical analyses, where applicable. Correlations were calculated using non-linear regression analyses. Supplementary figures.Click here for additional data file.