Healthcare Technology Letters (HTL) journal dedicated to the 12th edition of the Joint International Workshop on Augmented Environments for Computer-Assisted Interventions (AE-CAI) and Cerebral Visualization (CereVis). We are pleased to present the proceedings of this exciting scientific gathering held in conjunction with the Medical Image Computing and Computer-Assisted Interventions (MICCAI) conference on September 16th, 2018 in Granada, Spain.Welcome to this Special Issue of the IET's The AE-CAI workshop series dates as far back as MICCAI 2006 in Denmark and has evolved as a forum to discuss the latest developments in Computer-Assisted Interventions (CAI). CAI is a field of research and practice, where medical interventions are supported by computer-based tools and methodologies. CAI systems enable more precise, safer, and less invasive interventional treatments by providing enhanced planning, real-time visualization, instrument guidance and navigation, as well as situation awareness and cognition. These research domains have been motivated by the development of medical imaging and its evolution from being primarily a diagnostic modality towards its use as a therapeutic and interventional aid, driven by the need to streamline the diagnostic and therapeutic processes via minimally invasive visualization and therapy.To promote this field of research, the AE-CAI workshop series seeks to showcase papers that disseminate novel theoretical algorithms, technical implementations, and development and validation of integrated hardware and software systems in the context of their dedicated clinical applications. This year's workshop is a joint effort between two communities – the AE-CAI community and the CereVis community. We joined forces back in June 2018 in light of our common interests in neuro-navigation and visualization for image-guided neuro-interventions.The objective of the AE-CAI & CereVis workshop has been to attract scientific contributions that offer solutions to the technical problems in the area of augmented and virtual environments for computer-assisted interventions, with CereVis contributions focusing on neuro-navigation and neuro-interventions, and to provide a venue for dissemination of papers describing both complete systems and clinical applications. The workshop attracts researchers in computer science, biomedical engineering, computer vision, robotics, and medical imaging.The workshop featured a single track that consisted of long and short oral presentations showcasing original research engaged in the development of virtual and augmented environments for medical image visualization and image-guided interventions. To foster networking and discussion, we also provided all authors with the opportunity to present their work as a poster in addition to their oral presentation slot.In addition to the contributed papers, we were pleased to welcome two keynote speakers: Dr. Klaus Engel and Dr. Terry Peters. Dr. Engel is an expert in visualization from Siemens Healthineers in Erlangen, Germany and spoke on cinematic rendering from medical imaging. Dr. Terry Peters is a pioneer in image-guided interventions from Robarts Research Institute at Western University in London, Ontario, Canada and spoke on current and future trends in mixed, augmented and virtual reality visualization in medicine.Healthcare Technology Letters journal. This year AE-CAI & CereVis received 20 manuscripts spanning a strong geographic representation from Europe, North America and Asia. The review process was rigorous and involved evaluation of each manuscript by three to five external reviewers. Following the first stage of review, sixteen manuscripts were further considered for major or minor revision, and resubmission. Authors were required to submit a response to reviewers and resubmit a revised manuscript that addressed all reviewers’ critiques.All manuscripts submitted to the joint AE-CAI & CereVis 2018 were held up to journal standards, as the ultimate objective was to publish accepted work in this Special Issue of the IET's Once revised, all resubmitted manuscripts entered a second round of review conducted by the AE-CAI Program Committee, Workshop Chairs, as well as the HTL Managing Editor and Editor-in-Chief to ensure that all reviewers’ critiques were properly addressed in the revised manuscripts and that the quality of the manuscript was appropriate for journal publication. All manuscript were then accepted for publication and forwarded to the journal for production and publication.On behalf of the AE-CAI & CereVis 2018 Program and Organizing Committee, we would like to extend our sincere gratitude to all authors, presenters, and attendees for their scientific contribution, enthusiasm, and support. We also extend special thanks to all reviewers for providing detailed and timely critiques of the submitted manuscripts. We also acknowledge the support we received from the MICCAI 2018 Workshop Committee, and the HTL Editorial Office for their dedication to maintaining the high quality of AE-CAI and CereVis workshops, and for selecting outstanding manuscripts that fostered exciting discussions at the meeting.Northern Digital Inc. (NDI) for their continued support over the years that has enabled us to recognize AE-CAI authors for their much deserved dedication and scientific enthusiasm through several paper awards. We also thank BrainLab for their generous sponsorship of the top papers presented as part of the neuro-imaging/navigation/visualization track encompassed by the CereVis workshop. A big thank you on behalf of all awardees and once again, sincere congratulations to all award winners!Last but not least, we acknowledge our generous sponsors. We thank CereVis Award Papers:by David ShattuckA Multiuser Virtual Reality Environment for Visualizing Neuroimaging Data, by Taylor Frantz; Bart Jansen; Johnny Duerinck; Jef VandemeulebrouckeAugmenting Microsoft's HoloLens with Vuforia Tracking for Neuronavigation, by Reid Vassallo; Hidetoshi Kasuya; Benjamin Lo; Terry Peters; Yiming XiaoAugmented reality guidance in cerebrovascular surgery using microscopic video enhancement, AE-CAI Award Papers:by Gavin Wheeler; Shujie Deng; Nicolas Toussaint; Kuberan Pushparajah; Julia Schnabel; John Simpson; Alberto Gomez HerreroVirtual Interaction and Visualisation of 3D Medical Imaging Data with VTK and Unity, by Long Qian; Anton Deguet; Peter KazanzidesARssist: Augmented Reality on a Head-Mounted Display for the First Assistant in Robotic Surgery, by Ehsan Azimi; Tianyu Song; Chenglin Yang; Omid DianatEndodontic Guided Treatment Using Augmented Reality on a Head-Mounted Display System, by Andrew Speers; Burton Ma; William Jarnagin; Sharifa Himidan; Amber Simpson; Richard WildesFast and accurate vision-based stereo reconstruction and motion estimation for image-guided liver surgery, We hope that you will enjoy reading this Special Issue and we look forward to your continuing support and participation in future editions of the AE-CAI & CereVis workshops. Their continued success demands our ongoing commitment.Cristian A. Linte, Rochester Institute of Technology, USAMarta Kersten-Oertel, Concordia University, CanadaZiv Yaniv, National Institutes of Health and TAJ Technologies, USAYiming Xiao, Western University, CanadaCaroline Essert, University of Strasbourg, FrancePierre Jannin, University of Rennes IJonathan Lau, Western University, CanadaPhilip Pratt, Imperial College London, UKIngerid Reinertsen, SINTEF, NorwayHassan Rivaz, Concordia University, Canada


Drosophila pupal development, attenuated JNK signaling in the thoracic dorsal epithelium leads to defective midline closure, resulting in cleft thorax.Downregulation of Jun N-terminal kinase (JNK) signaling inhibits cell migration in diverse model systems. In  Drosophila pupal development, attenuated JNK signaling in the thoracic dorsal epithelium leads to defective midline closure, resulting in cleft thorax. Here we report that concomitant expression of the Ciona intestinalis alternative oxidase (AOX) was able to compensate for JNK pathway downregulation, substantially correcting the cleft thorax phenotype. AOX expression also promoted wound-healing behavior and single-cell migration in immortalized mouse embryonic fibroblasts (iMEFs), counteracting the effect of JNK pathway inhibition. However, AOX was not able to rescue developmental phenotypes resulting from knockdown of the AP-1 transcription factor, the canonical target of JNK, nor its targets and had no effect on AP-1-dependent transcription. The migration of AOX-expressing iMEFs in the wound-healing assay was differentially stimulated by antimycin A, which redirects respiratory electron flow through AOX, altering the balance between mitochondrial ATP and heat production. Since other treatments affecting mitochondrial ATP did not stimulate wound healing, we propose increased mitochondrial heat production as the most likely primary mechanism of action of AOX in promoting cell migration in these various contexts.Downregulation of Jun N-terminal kinase (JNK) signaling inhibits cell migration in diverse model systems. In  Drosophila development, cell migration has been studied in embryogenesis, in the process of dorsal closure  from Ciona AOX can alleviate the damaging phenotypes associated with RC inhibition , which is found in microbes, plants, and some metazoan phyla but not insects or vertebrates . AOX prohibition –19. HoweDrosophila RXR homologue, ultraspiracle (usp), which acts as a dimerization partner for the ecdysone receptor       signaling pathway  Fig. 1AFig. 1A. us (hep)  or of thn c-Fos) . The userotease) , or overck loop)  or the ts (Timp)  can alsos of hep . One keys of hep , 36 is ting edge , but it ing edge . One keye is pnr . A dpp h closure .We therefore set out to test whether AOX could rescue cleft thorax when induced by manipulations of the JNK pathway and the associated gene network described above.pnr-GAL4 (MD237pnr) driver, that the downregulation of key components of the JNK signaling cascade , hemipterous , misshapen , and PDGF- and VEGF-receptor related . These all produced a cleft thorax phenotype of various severities , we then combined these with expression constructs for AOX or for a control transgene, the GFP gene  and the  cascade  22) in  in pnr-GGFP gene  and 3.bsk and hep we tested multiple RNAi lines (Table S1), each producing cleft thorax when combined with the pnr-GAL4 driver. Although the severity of cleft thorax varied slightly between experiments, expression of AOX (bsk or hep knockdown flies. Two different RNAi lines for each gene showed the same effect (pnr-GAL4 in pupae with and without one of the double-stranded RNA (dsRNA) constructs for hep, which showed no significant difference  were isolated from AOX hemizygous mice and wild-type littermates and immortalized using a standard retroviral transduction procedure with viruses encoding human papillomavirus 16 (HPV16) oncoproteins E6 and E7  and kayak (kay), respectively, should produce effects that largely override its regulation by JNK and the beneficial effects of AOX. Accordingly, coexpression of AOX had only a slight effect on the severity of cleft thorax induced by knockdown of kay or Jra using the pnr-GAL4 driver , to which AP-1 does not bind.To investigate the mechanism by which AOX impacts the outcome of JNK signaling, we tested whether it influences transcription directed by AP-1. Using a well-established luciferase-based AP-1 reporter system  and a vahep had been knocked down , stably transduced with lentiviral constructs expressing AOX or, as a control, the mutated, catalytically inactive variant (mutAOX). Successful transduction and cell cloning at limiting dilution were verified via the fluorescence conferred by the cotransduced marker GFP, and AOX functionality was verified by respirometry (Table S5). Although individual HEK-AP1 cell-derived clones showed a variable degree of AP-1-dependent transcriptional activity, AOX-expressing and control cell clones showed a similar susceptibility to the effects of the JNK antagonists SP600125 and inhibitor V . Surprisp-trifluoromethoxyphenylhydrazone [FCCP]), and measured wound closure in four independent experiments. Antimycin A had a significantly different effect on the migration of AOX-expressing MEFs versus wild-type MEFs (To gain insight into the intracellular process(es) underlying the enhanced migratory behavior of AOX-expressing cells, we tested the effects of sublethal doses of various metabolic effectors on the relative rates of migration of AOX-expressing versus control iMEFs. In an initial experiment , we testype MEFs , stimulaype MEFs . AOX hadDrosophila, AOX expression corrected thoracic closure defects produced by impaired signaling at several steps in the JNK pathway  or mammalian (HEK293-derived) cells. The migration of AOX-expressing iMEFs compared with that of control iMEFs was differentially stimulated by antimycin A but not by other drugs that affect mitochondrial electron flow, ATP production, reactive oxygen species (ROS), or membrane potential. We thus propose increased mitochondrial heat production as the most likely mechanism by which AOX promotes cell migration.In this study, we showed that the expression of AOX is able to promote cell migratory behavior in two different models: in Clearly, to affect cell motility or any other phenotype, AOX needs to be enzymatically active, modifying the cellular metabolic state. Previous studies in model systems –19 found–The proteins used for immortalization of MEFs, namely, the HPV16 E6 and E7 oncoproteins, may also synergize with activated AOX. Among other effects , E7 swit55–59–Drosophila findings and the effects of inhibitor V leave open a possible direct involvement of JNK, acting through other targets.Reporter assays indicate that AP-1 and its transcriptional activity are not a direct target of AOX, although the The transcriptional reporter cell assays were conducted in proliferating cells, where AP-1 may act differently than in a migrating cell sheet. It plays diverse physiological roles, and each of its subunits is encoded by a gene family . Althougin vitro systems -fluoromethylketone (Z-VAD-FMK)  or decrcysteine , MitoQ  also apSince AOX-expressing iMEFs continue to respire when treated with antimycin A, most metabolites should be only minimally affected. The biggest difference should be in the rate of mitochondrial ATP production, when respiratory complexes III and IV are replaced by the non-proton-motive AOX. However, as indicated above, mitochondrial ATP production as such cannot be the crucial factor determining the rate of migration.In the presence of antimycin A, the energy released by AOX in catalyzing quinol oxidation is instead converted to heat. We earlier showed that AOX-driven respiration in human cells was able to maintain the same mitochondrial temperature with less than half the amount of respiratory flux . In the Fibroblast motility has long been known to be temperature dependent , and actTogether, these data strongly suggest that AOX activation is able to promote cell migration by raising the intracellular temperature, most likely in specific subcellular compartments. The compensatory effects on JNK signaling are thus implied to be indirect, accounting also for the fact that AOX was able to correct cleft thorax in a completely different model generated by deranged nuclear receptor signaling .in situ. It will require even more sophisticated technology to make such measurements in vivo in the Drosophila pupa. Alternatively, if some other metabolic effect of AOX-driven respiration is responsible, we would need to wait for metabolomic technologies to advance to the single-cell level  supplemented with 20% (primary MEFs) or 10% (iMEFs) heat-inactivated fetal bovine serum , 1% penicillin-streptomycin (Lonza), and 4 mM l-glutamine (Sigma-Aldrich). An AP-1 reporter HEK293 recombinant cell line , here abbreviated HEK-AP1, was maintained at 37°C in 5% CO2 in minimal essential medium (HyClone) supplemented with 10% heat-inactivated FBS (Sigma-Aldrich), 1% nonessential amino acids (HyClone), 1 mM sodium pyruvate (Sigma-Aldrich), 1% penicillin-streptomycin (Lonza), and 400 μg/ml Geneticin . Geneticin was freshly distributed to each plate when cells were passaged. BJ-5ta human fibroblasts (ATCC CRL-4001) were grown at 37°C in 5% CO2 in DMEM (Sigma-Aldrich) supplemented with 10% heat-inactivated FBS (Sigma-Aldrich), 20% medium 199 (Sigma-Aldrich), 1% penicillin-streptomycin (Lonza), and 4 mM l-glutamine (Sigma-Aldrich). HEK-AP1 and BJ-5ta cells expressing C. intestinalis AOX or the mutated, catalytically inactive variant mutAOX , chloroquine (20 μM), E64d (10 μg/ml), MitoQ , valinomycin (10 μM), Z-VAD-FMK (20 μM), or just 4 μl DMSO as a solvent control. All chemicals were from Sigma-Aldrich, except where stated otherwise. To measure single-cell migration, samples of 15,000 cells were plated on 6-well plates  as technical triplicates (3 wells per sample). To minimize plate-specific effects, each plate contained both AOX-positive and -negative iMEFs in all assays. Cells were imaged as described below.Samples of 90,000 cells were plated on 24-well plates  as technical triplicates (3 wells per sample). After 24 h, cells were treated with one of the following reagents: 0.2% dimethyl sulfoxide , 20 μM SP600125 (Sigma-Aldrich) in 0.2% DMSO, 20 μM JNK inhibitor V (Merck), or 20 nM phorbol myristate acetate  in fresh medium. After 2 h, a linear scratch was made in the center of each well with a p10 (1- to 10-μl) pipette tip. The cells were then washed three times with medium to remove detached cells, and fresh medium containing the appropriate reagent was added to each well. For testing the effects of metabolic inhibitors in this assay, cells were seeded at 45,000 to 90,000 cells per well on 24-well plates until a monolayer was formed (24 to 48 h), before the scratch was made. After rinsing once with normal medium, medium was replaced with 1 ml of fresh medium containing one of the following drugs: oligomycin (1 ng/ml), antimycin A (60 ng/ml), rotenone (150 nM), FCCP (10 μM), Drosophila embryos, eggs were collected from grape juice-agar plates after adult flies were placed in a mating chamber for 1 h, covered by a dark box, dechorionated using double-sided tape  by manually tracking the gap area, based on lines drawn along the wounded area of an image taken every second hour. The speed of the collective motion of the cells was measured in the most linear part of the wound area over time. In the experiment using metabolic inhibitors, the most linear time interval for closure was also analyzed and applied across all treatments and plates. To generate images for measuring single-cell migration, cells were transferred 24 h after plating to a Nikon BioStation CT instrument equipped with a Nikon DS-1QM camera and imaged at ×4 magnification. The environment in the incubator was held at a temperature of 37°C with a relative humidity of 85% and a 5% CO2 atmosphere, as described above for the wound-healing assay. Images were taken every 6 min, and movies were made using CL Quant (version 3.10) software. Movement was analyzed using CellTracker image-processing software with semiautomated migration detection  software. For live imaging of ded tape , placed etection , ignorinhep mRNA were TTGGTTTCTTGGGGTCGATG and TGGACTCCAAGGCCAACACT, the sequences of both of which are shown 5′ to 3′.RNA was extracted from S2 cells , and quaact, kindly supplied by Dirk Bohmann, University of Rochester. Transfections used either TRE-fluc (a firefly luciferase reporter with the intact phorbol-ester response element) or mRE-fluc (with the mutated response element) plus pAct-RL  with or without pAct-Gal4 together with pUAST-Hepact for activated transcription and with the following plasmids or controls harboring AOX, as indicated in the figure legends: pMT-AOX , containing the C. intestinalis AOX-coding sequence recloned with its natural stop codon from the original vector, pCDNA5/FRT/TO ; and pUASTattB-AOX and pUASTattB-mutAOX , and sterile water up to a final volume of 100 μl (6-well plates) or 50 μl (12-well plates). Transfections were carried out 1 h after plating , and luciferase assays were performed 72 h after transfection. In transfection mixtures to which mitochondrial inhibitors were added, 6 × 105 cells/ml were plated on 6-well plates, and drugs were added 24 h after transfection at the concentrations shown in the figure legends. For luciferase reporter experiments in which hep expression was knocked down by RNAi, cells were also transfected with a dsRNA against the hep coding sequence, prepared by a two-round PCR-based procedure essentially as described previously (hep-specific primers (shown 5′ to 3′) TGGAGGCAAAGCTCCAGGC and CGCGAACGAAGCAGCCAAGG for the first round and GAATTAATACGACTCACTATAGGGGAGACATCCGCCACCCACGCACCTTC and GAATTAATACGACTCACTATAGGGGAGATCCCATTGCCCAGGTCGCCCAG for the second round, followed by transcription using a MEGAscript T7 transcription kit (Life Technologies). Knockdown at the RNA level (to ∼85%) was verified in transfected cells  and analyzed using a Dual-Glo luciferase assay system (Promega), according to the manufacturer's protocol. Luminescence was measured using a Thermo Labsystems Luminoskan Ascent plate reader.Firefly/renilla luciferase dual-reporter assays in S2 cells used a cotransfection protocol , based o5/FRT/TO , as an E5/FRT/TO , 112, coB-mutAOX , containB-mutAOX , prior tFirefly luciferase reporter assays were carried out in HEK-AP1 cells and in AOX/mutAOX-expressing clones derived from them, as follows: 30,000 cells were plated in technical duplicate (2 wells per sample) in luminometer-compatible Nunc MicroWell 96-well plates with lids (Thermo Fisher Scientific). After 24 h, the medium was replaced with medium containing either 0.2% DMSO, 20 μM SP600125 in 0.2% DMSO, 20 μM JNK inhibitor V, or no added drug. Cells were incubated for 2 h at 37°C. For PMA treatment, a second replacement medium contained 8 nM PMA plus 20 μM SP600125 in 0.2% DMSO, 20 μM JNK inhibitor V, or no other added drug, as appropriate, and the cells were incubated for a further 6 h. Luciferase assays were carried out using the Dual-Glo luciferase assay system (Promega), according to the manufacturer's protocol, and luminescence was measured using a PerkinElmer UV/visible plate reader.Batches of 300,000 MEFs or HEK-AP1 cells or 250,000 BJ-5ta cells were plated on 6-well plates . After 24 h, the medium was replaced with medium containing either 0.2% DMSO, 20 μM SP600125 in 0.2% DMSO, 20 μM JNK inhibitor V, 8 nM PMA, or no added drug and the plate was incubated for 2 h . Cells were carefully rinsed in ice-cold phosphate-buffered saline (PBS) and then scraped free on ice using a CytOne cell scraper  in 75 μl of resuspension buffer containing 100 mM NaCl, 10 mM Tris-HCl, and 1 mM EDTA, pH 7.8, supplemented with cOmplete, Mini, EDTA-free protease inhibitor and phosphatase inhibitor cocktails  and 1 mM phenylmethylsulfonyl fluoride. Protein concentrations were determined using the Bradford assay. After lysis by the addition of an equal volume of SDS sample buffer , samples were heated for 5 min at 100°C and briefly centrifuged to remove particulates, and 20 μg of each extract was loaded onto 18-well precast Any kD Criterion TGX Stain-Free protein gels (Bio-Rad), which were run and blotted as described previously . Blots w6 iMEFs being directly suspended in the oxygraph chamber containing 2 ml of respiration buffer B . After measuring endogenous whole-cell respiration, substrates and inhibitors were added in the following order: (i) digitonin (30 μg), to permeabilize the cells; (ii) sodium pyruvate (to 5 mM), sodium glutamate (to 5 mM), and sodium malate (to 2 mM) as a cI-linked substrate mix, followed by ADP (to 2 mM); (iii) rotenone (to 150 nM) followed by succinate (to 10 mM) as a cII-linked substrate mix; (iv) antimycin A (to 30 ng/ml), to reveal AOX-mediated respiration; (v) n-propyl gallate , to reveal any residual non-AOX-mediated oxygen consumption to be subtracted; (vi) N,N,N′,N′-tetramethyl-p-phenylenediamine  plus sodium l-ascorbate (to 2 mM) as a cIV-linked substrate mix; and (vii) sodium azide (to 40 mM), to reveal any non-cIV-mediated oxygen consumption to be subtracted. O2 consumption (in picomoles · second−1 · milliliter−1) was normalized to the amount of total proteins extracted from 1 × 106 cells and assayed by the Bradford method (Whole-cell and permeabilized cell respiration was measured essentially as described previously . iMEFs wd method . All che


Human T-lymphotropic virus type 1 (HTLV-1) and other members of the Deltaretrovirus genus code for a regulatory protein named Rex that binds to the Rex-responsive element present on viral mRNAs. Rex rescues viral mRNAs from complete splicing or degradation and guides them to the cytoplasm for translation. The activity of Rex is essential for expression of viral transcripts coding for the virion components and thus represents a potential target for virus eradication. We present an overview of the functional properties of the HTLV-1 and HTLV-2 Rex proteins (Rex-1 and Rex-2), outline mechanisms controlling Rex function, and discuss similarities and differences in the sequences of Rex coded by HTLV-1, -2, -3, and -4 that may influence their molecular anatomy and functional properties. Human T-lymphotropic virus type 1 (HTLV-1) infects approximately 10 million persons worldwide . HTLV-1 The replication cycle of the HTLVs  is controlled by the viral regulatory proteins Tax and Rex, which are coded in open reading frames (ORFs) named x-IV and x-III, respectively, located on the plus-strand of the proviral genome. Tax increases transcription from the 5′LTR promoter as well as the promoters of many cellular genes, and plays a key role in viral replication and cell transformation . As illuThe RXRE is located in the 3′LTR, with RXRE-1 mapping to a short portion of U3 and most of R  and RXREin vitro experiments suggest that Rex-1 may also interfere with the activity of Dicer  machinery. A study of HTLV-1-infected cells provided evidence that Rex contributes to suppress NMD of viral and cellular transcripts through a mechanism that does not involve interaction with the RXRE . Resultsof Dicer , a key cStudies of Rex and its HIV homolog Rev have contributed substantially to our knowledge of RNA processing as well as nucleo-cytoplasmic shuttling of proteins and RNA . The folAlmost all of the information on Rex was gathered from studies of Rex-1 coded by subtype-a isolate ATK-1 and Rex-2 coded by subtype-a isolate Mo. Rex-1 (ATK-1) and Rex-2 (Mo) contain 189- and 170 amino acids, respectively, and four main functional domains . An aminRex-1 proteins coded by isolates of subtypes a, b, and c also showed some variation, especially between subtype-a and subtype-c ORFs   ORF produces two isoforms of 24- and 26 kDa that differ in phosphorylation . Mass spThe protein kinases responsible for Rex phosphorylation have not been experimentally identified. Analysis of the Rex-1 (ATK-1) and Rex-2 (Mo) ORFs with the NetPhos and ScanSite prediction tools yielded one or more candidate kinases for most of the phosphoacceptor sites identified by mass spectrometry , b as wein vivo – predominantly CD4 + T-cells for HTLV-1  transfected with Rex expression plasmids. As the expression of protein kinases can be highly cell-type specific, it will be important to study the protein in cells that are natural targets of HTLV infection r HTLV-1  and CD8 r HTLV-2 . The ider HTLV-2 .As depicted in In HTLV-2, spliced mRNAs 1-3 and 1-B code for truncated Rex-2 proteins named p22/p20Rex and p17Rex . These mWhile Met-33 is not present in any of the 15 Rex-1 proteins or in Rex-3 shown in b (214 a.a.) and Rexc (213 a.a.), respectively, but do not produce Tax, and mRNA 1-2-3a produces a 185-residue Rex-1 isoform named Rexa and a Tax isoform lacking four amino-proximal amino acids. Results of functional assays showed that all three Rex isoforms are functionally active. However, Rexb and Rexc are mainly cytoplasmic, suggesting that their extra amino acids affect the NLS and/or NES  located upstream  or downstream (3a) of the canonical exon 3 SA; the use of these SA results in insertion or deletion of amino acids in the Rex ORF just after the NLS/RBD . Transcrd/or NES . It willa but not Rexb or Rexc. Among the HTLV-2, HTLV-3, and HTLV-4 isolates in a-like protein but not Rexb or Rexc due to the absence of the corresponding SAs and presence of one or more stop codons. HTLV-2b isolate Gu lacks all three alternative SAs and contains stop codons, and thus should not produce any of the extra Rex isoforms.The possibility that the other HTLVs produce analogous Rex isoforms remains to be investigated. SAs C, Ca and 3a are present in all 15 HTLV-1 isolates listed in The first investigation of HTLV-1 mRNA expression kinetics, performed by transfecting a full-length HTLV-1 provirus and northern blotting, revealed early (Rex-independent) expression of multiply spliced mRNA and late (Rex-dependent) accumulation of singly spliced and unspliced mRNA . This twb and Rexc but not Tax.Mathematical modeling of HTLV-1 expression indicated the requirement for a delay in Rex function compared with Tax in order to support the 2-phase kinetics observed experimentally . HoweverIt is also possible that changes in the availability of cellular factors influence the relative activity of the 2 proteins during the expression cycle. An example is heterogenous nuclear ribonucleoprotein A1 (hnRNP A1), an important regulator of RNA processing that was shown to interfere with Rex function by competitively binding to the RXRE  Figure .Recent studies indicated that individual HTLV-1-infected cells undergo alternating bursts of viral gene expression and latency . Shut-doStudies of HTLV-2 expression kinetics indicated a similar 2-phase pattern of mRNA production . The obsin vitro immortalization of cultured T-cells , it is required for establishment of persistent infection in a rabbit model (Comparisons of the biological properties of wild-type and Rex-defective HTLV-1 molecular clones indicated that while Rex is not essential for it model . This isit model .The discovery of Rex-1 as an essential factor for expression of Gag/Pro/Pol RNA  opened uAll authors worked together to prepare the manuscript. DD performed the sequence alignments and prepared the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In 2001, the CORE Group Polio Project (CGPP) began to support polio eradication initiatives in hard-to-reach pastoralist and semi-pastoralist high-risk border areas of Ethiopia by training and supporting community volunteers (CVs) for immunization promotion and community-based surveillance activities. This article describes the development and current status of the CGPP CV network in Ethiopia. It also reports the results of a 2016 survey of CVs. Community volunteers are selected jointly by the local community, local government officials, and local health facility staff. They work closely with the health extension worker in their area and are responsible for 50–100 households. More than 12,000 CVs have been trained and have reached six million people. They make routine home visits to 1) provide education on vaccine-preventable diseases, 2) promote healthy behaviors, 3) inform parents on how to access immunization services, and 4) report cases of acute flaccid paralysis, neonatal tetanus, and measles as well as births. The 2016 survey of 675 CVs demonstrated that 84.1% had conducted home visits in the previous month to 1) identify and register pregnant mothers and newborns, 2) provide health education, 3) conduct disease surveillance, and 4) search for and register immunization defaulters. Of the CVs, 98.2% reported that their work had led to improvements in the community. Knowledge of CVs about vaccine-preventable diseases was suboptimal. CVs expressed a desire for more training. Community volunteers have made notable contributions to polio eradication efforts in high-risk areas of Ethiopia as well as to immunization promotion and disease control more broadly. More than 10 million community-based health volunteers have supported supplemental immunization activities communities and administering oral polio vaccine.4Community-based health volunteers represent an important resource for health programs throughout the world, particularly in resource-constrained settings.5 assesses the improvements in routine polio immunization coverage in the areas where these CGPP CVs have been active. A history of the CGPP’s global activities and its legacy are presented elsewhere in this series.7This article describes the development, knowledge, and practices of a specialized type of community volunteers (CVs) in Ethiopia who have been trained for a highly specific ongoing role in high-priority areas where polio immunization coverage is low, where the risk of active transmission of poliovirus is high, and where there is a need for stronger surveillance. The CORE Group Polio Project (CGPP) has trained and deployed CVs in sparsely populated regions of Ethiopia inhabited by pastoralist and semi-pastoralist populations and in border areas at high risk for transmission of poliovirus from neighboring countries. Included in this article are the findings of a 2016 survey of CVs. Another article in this seriesThe CGPP has been working in Ethiopia since 2001 in pastoralist and semi-pastoralist areas and in high-risk border areas. In these areas, high levels of immunization coverage have been difficult to achieve. Wild poliovirus transmission persisted until 2014, when the last case of wild poliovirus was identified. The final cases of polio occurred in areas where levels of immunization coverage were low or where levels of immunization across the national border and the borders were porous and, therefore, where the risk of cross-border transmission was high.kebele, or sub-district, with a population of approximately 5,000 people) and local health facilities are also involved in the selection of CGPP CVs. The criteria for selection of CGPP CVs include having interest in delivering health messages and health care to the local community, being well accepted by the community, being a good role model, speaking the local language, and living in the community.The CGPP CVs are nominated by their respective communities. In addition, the lowest level of government administration .Once selected, the CGPP CV begins to work with the health extension worker (HEW), a government paid, full-time community health worker (described further later). There are about three to five CGPP CVs per 6 The training is based on a standardized training manual produced by the CGPP in Amharic and English as well as in local languages such as Somali and Oromifa. The training manual is distributed to all CVs and serves as a reference for all CVs, many of whom are illiterate. Literate family members assist illiterate CVs when necessary.The CGPP provides its CVs with 3 days of training on immunization, vaccine-preventable diseases, and community-based surveillance. The training is provided by field officers of non-governmental organizations (NGOs) that are CGPP project implementation partners as well as by HEWs. The CGPP implements its work through a secretariat, which engages international and local NGOs to take responsibility for CGPP activities in specific geographic areas.CVs are active in the areas where the CGPP is active. They exist in all 85 hard-to-reach pastoralist, semi-pastoralist, and international border areas of CGPP implementation districts of five regions of the country: Benishangul-Gumuz; Gambella; Oromia; Somali and Southern Nations, Nationalities, and Peoples (SNNP) Regions. CVs visit all households to provide education on vaccine-preventable diseases and on how to access immunization services. They also actively search for and report cases of the three types of diseases under surveillance: acute flaccid paralysis (AFP), measles, and tetanus. In addition, they identify newly pregnant women, newborns, and those in need of immunizations. The CGPP CVs provide oral or written reports to their supervising HEW, who then sends this information to the next higher level. The CGPP expects CVs to work 4 hours a week (2 hours per day for 2 days each week) at a minimum. CVs draft monthly plans and are supervised by HEWs to ensure that all households are reached within the 3-month reporting period. The order of visits is decided by the CVs. In addition, CVs make visits based on real-time information, such as new births in their communities or suspected AFP cases. They are supposed to visit four households per day (or eight households per week), spending half an hour with each household, and also to give priority to villages that are located farthest away.woreda (district) health office. Feedback from the supervision is given to supervisees and also to other stakeholders. Individual supervision is also provided by HEWs. CGPP/Ethiopia secretariat staff members conduct quarterly joint supportive supervision visits to his/her assigned areas during each supervision period, at which time they visit two woredas, one health center, two health posts, and three CGPP CVs in each woreda.Supportive supervision is a facilitative approach to supervision that promotes mentorship, joint problem-solving, and communication between supervisors and supervisees. Effective supportive supervision is critical for creating an enabling work environment, maintaining a well-functioning system, and motivating supervisees. Supportive supervision is provided for the CGPP CVs, both jointly and individually. Regular periodic joint supportive supervision using a standardized checklist is conducted by individual CGPP secretariat members, staff members from the CGPP Project NGO implementing partners, and the woreda health office level where all the CGPP CVs, Health Development Army (HDA) volunteers (described later), HEWs, and woreda health officials meet. These meetings often have 100 participants. The monthly meetings take 1 day, and the quarterly meetings take 2–3 days. In addition to their regular meetings, CVs meet periodically with their supervising HEW, and together they exchange ideas and conduct house-to-house visits.The CGPP CVs attend regular monthly meetings organized at the health post to which they are attached (and where two HEWs are based). At the time of these meetings, the CVs submit their monthly report, jointly review progress in the catchment area, prepare a work plan for the coming month, discuss constraints and solutions, and update their knowledge on surveillance and immunization. In addition, quarterly meetings are held at the woreda staff, the community’s acceptance of messages transmitted by the CGPP CVs, and opportunities to learn new and useful information.8 As an additional incentive, the CGPP provides its CVs with an umbrella, an apron, a bag, and a coat.CVs are motivated by feelings of responsibility, trust, acceptance, and recognition by their communities to serve and promote immunization and health. Community volunteers are not paid but receive small non-financial incentives to motivate them. Together, the community, the CGPP secretariat, and the NGO partners provide a certificate of recognition for CVs, and the best performing CVs receive an award such as a mobile phone or radio. Other motivating influences include the supportive supervision and job aids provided by HEWs and 9 Similar activities spread throughout Ethiopia and led to the utilization of CVs by many NGOs and by vertical government programs such as family planning and HIV control.In the 1970s, community-based activities for maternal, child, and environmental health programs  in the Tigray regional state of northern Ethiopia began to use CVs.11 These programs aimed to expand the health service coverage in all regions of Ethiopia, especially in rural parts of the country. The Health Extension Program is a comprehensive set of preventive, promotive, and curative services that have been combined as 17 separate packages, as shown in In 2003, Ethiopia’s Federal Ministry of Health launched a new health-care plan, called the Accelerated Expansion of Primary Health Care Coverage, as part of a comprehensive Health Extension Program. The Health Extension Program represented an innovative, community-driven approach whose goal was the universal coverage of primary health care.kebele . In 2012, the government of Ethiopia introduced the HDA, which replaced most CV programs in health that had been developed by the government and NGOs for various purposes.12 Although the intention of government is to eventually replace all CVs with HDA volunteers, the CGPP CVs are still functioning in the CGPP implementation areas. In the future, many of the trained CGPP CVs will be absorbed into the HDA, and it is the government’s vision that the HDA will carry out polio eradication activities in the CGPP implementation areas where the HDA program is now not well-developed . Trained informally by HEWs and working under their direct supervision, HDA volunteers provide health education and support social mobilization for health. They also serve as guides for HEWs when these workers come for community-level activities.Beginning in 2004, the Ethiopian government provided 1 year of training and deployed 38,000 full-time, salaried female HEWs to work at health posts, with two HEWs per health post and one health post serving a 12 Nationwide, there are now approximately three million HDA volunteers.12 In the CGPP implementation areas where HDA volunteers are now active, the CGPP has trained HDA leaders (of which there is one for every 30 HDA volunteers) with the same training provided to CVs, and they take on the same tasks as CVs.The HDA program is based on the identification and training of “model families” . In practice, “model families” become leaders of a group of five families known as the “one-to-five network,” and these, in turn, combined to form a group of 25–30 “model households” within a village. Women who are living in “model households” become eligible to become an HDA volunteer after finishing 96 hours of training on the Health Extension Program and implementing model behaviors in their household.woreda health offices. The training takes 15 days on average, and it focuses on high-impact maternal, newborn, and child health measures. In the training, in a kebele of 1,000 households , 150 HDA volunteers on average are expected to attend. After the introduction of the HDA, some of those who had been previously serving as a CGPP CV also became HDA volunteers. The HDA was intended to replace most other CV programs that had been developed by either the government or by NGOs before the introduction of the HDA in 2012.HDA volunteers collaborating with the CGPP take on the same responsibilities as CVs for polio eradication, promotion of routine immunization, and surveillance, as well as in supporting HEWs more generally in the implementation of the Health Extension Program. There is no literacy requirement to become an HDA volunteer. The informal training of HDA volunteers is facilitated by HEWs with support from primary health care units and oreda, the local kebeles, and HEWs. By contrast, the HDA has a tight governance structure; it is led by a command post established at each level of the government’s administrative structure: region, zone, district, and kebele. Just as for the CGPP CVs, there are no literacy requirements for HDA volunteers. However, in contrast to CGPP CVs, who are both men and women, all HDA volunteers, are women.The CGPP CVs are selected by the community together with the community leaders, the wIn areas where the CGPP is functioning, there is one CGPP CV for every 100–200 households, although there is an HDA volunteer for every five households, and the HDA volunteer is supposed to come from a model household. Whereas CGPP CVs work on immunization and on surveillance of vaccine-preventable diseases, HDA volunteers are responsible for promoting the 17 packages of the Health Extension Program shown in . In addiworedas. At present, 11,650 CGPP CVs  make home visits, provide health promotion as described earlier, and look for cases of AFP, neonatal tetanus, and measles. In addition, they track the vaccination status of pregnant women and newborns.Since 2001, the CGPP has trained and placed more than 12,000 CVs along Ethiopia’s vulnerable borders with South Sudan, Kenya, and Somalia and in pastoralist/semi-pastoralist areas. As mentioned previously, the CGPP operates through a secretariat, hosted by the Consortium of Christian Relief and Development Associations, an Ethiopian NGO. The secretariat contracts with five international NGOs that contract with four national NGOs to implement the CGPP programs . The CGP13 In addition to their achievements in expanding polio vaccine coverage and caregiver knowledge about polio which is documented elsewhere in this series,5 the CGPP CVs during this 5-year period were able to double the percentage of fully immunized children (from 25% to 44% according to data recorded on immunization cards), to increase the detection rate of non-polio AFP cases from 2.2 to 2.8 per 100,000 children younger than 15 years per year , and to maintain an adequacy of stool samples from children with AFP at 87–92%.13 These achievements are notable given that the areas where the CGPP operates are in the most challenging and hardest-to-reach areas of the country.During the 5-year period between 2012 and 2017, the CGPP CVs identified and referred 306,432 women in need of tetanus immunization, 181,192 newborns in need of immunization, and 71,904 other children younger than 1 year of age who had fallen behind on their immunization schedule.Having now described the CGPP CV program, we now proceed to describe the findings from a recent survey of the CGPP CVs in Ethiopia.kebeles in five regions of the country: Somali; Oromia; SNNPR; Benishangul-Gumuz; and Gambella.At the time of the 2016 survey, there were 11,650 CGPP CVs and HDA volunteers working in 85 districts with 1,638 kebeles inhabited by pastoral and semi-pastoral people. Of the 723 kebeles in this geographic area, 223 kebeles were randomly selected. Within each of these selected kebeles, three CGPP CVs were randomly selected for interviews. One additional CV from each of six kebeles was interviewed, giving a total sample size 675.The survey consisted of 675 CVs of 2,857 CGPP CVs (excluding HDA volunteers) who were working in 11 zones†The study questionnaire was prepared in English and then translated into Somali, Oromifa, and Amharic languages. Face-to-face interviews were conducted in 2016 by data collectors who were community members with at least a high-school education and who were able to speak the local language in addition to Amharic. The data collectors received a 1-day training on the study objectives, selection of respondents, and interview procedures. The interviewers were supervised by the CGPP coordinators, temporary staff with experience in survey supervision, or government health officials.EpiData was used to enter the data with double entry verification. STATA version 13.0  was used for data analysis.woreda health offices, and the woreda health officer provided a letter to each of the kebele offices requesting permission and collaboration in conducting the survey. Data collectors, supervisors, and coordinators were trained on the objectives of the study, interviewing techniques, research ethics, and data quality issues. Interviewers obtained consent from all respondents, and participation was voluntary. Verbal consent was obtained from each participant before beginning the interview. All participants were assured of the confidentiality of their responses. Because this survey was carried out as part of the ongoing monitoring and evaluation of the CGPP Ethiopia program, it was not considered to be research and was not submitted to an ethical review board for approval.The CGPP Ethiopia secretariat office provided a letter to the respective regional, zonal, and Of the 675 respondents, 51.5% (348/675) were female, 88.3% (596/675) were married, 58.9% were aged 30–49 years, 30.6% (207/675) were younger than 30 years, 10.2% were aged 50 years or older, 26.6% had no formal education, 51.9% (350/675) had 1–8 years of schooling, and 21.5% had attended grade 9 or above.The distribution of CVs by region state was as follows: 45.6% were in Somali, 22.2% in Gambella, 19.1% (129/675) in Oromia, and 13.4% (90/675) in SNNPR. The regional state with the highest percentage of respondents who had no education was Somali (35.8%), whereas Oromia had the lowest (12.4%).kebele for more than 30 years, 58.5% had lived in the kebele for 10–29 years, and the rest (7.3%) had resided for less than 10 years. Of the respondents, 54.3% had served as a CGPP CV for 2–4 years, 32.3% had served in this role for five or more years, and 13.3% had served for fewer than 2 years. Of the respondents, 40.0% reported that they had been selected by the community and another 18.8% were selected by a committee composed of staff from the woreda health office, the primary health-care center, and the health post. The rest (41.1%) were selected by a combination of staff from health facilities, community leaders, and kebele administrators.Of the respondents, 34.2% had resided in their Among the 80.7% of the respondents who reported that they had received training provided by the CGPP, most (77.5%) received three or more days of training, whereas 12.4% reported fewer than 3 days of training and 14.2% received training for 7 days or more. Among the respondents with training, at least two-thirds had obtained training on the importance of immunizations, on registration of pregnant women and newborns, and on disease surveillance and reporting. Respondents reported that refresher training was given based on needs identified by supervisors. CVs sometimes leave abruptly and need to be replaced immediately. The replaced volunteers are sometimes not trained immediately and must be deployed without extensive training.As shown in Regarding the knowledge of CGPP CVs on signs and symptoms of AFP, 76.9% spontaneously mentioned limp limbs, 74.6% spontaneously mentioned cessation of walking or crawling as signs and symptoms of AFP, and 13.1% did not spontaneously mention any signs or symptoms. If a child with AFP was identified, 87.0% of CVs stated that they would accompany the parents and child to the nearest health facility, 34.4% said they would advise parents to do so, and 9.0% of CVs said that a child with AFP should be taken to a religious healer.Of the CGPP CVs, 84.1% reported that they had made home visits in the previous month. Among those who reported conducting home visits, 74.9% responded that they identified and registered pregnant mothers, 73.6% provided health education, and 58.3% identified and registered newborns. Of the respondents, 51.6% reported that they conducted disease surveillance , 42.6% stated that they searched for and registered immunization defaulters, and 41.6% stated that they carried out social mobilization activities. Of the CVS, 84.2% reported that they delivered health education to mothers about immunization during the postpartum period, at which time they also provided education to the mother about newborn care.Of the respondents, 83.7% reported that they regularly attend monthly meetings at the health post. The reasons cited for not attending included that the meetings were not held (43.7%), the respondents were too busy (43.7%), or that the meeting was too far away from home (15.5%). Of the respondents, 89.1% said that they submitted monthly reports. The reports were submitted to health posts in a majority of the (73.8%) cases.Of the respondents, 72.3% reported that they had a registration book in which they documented their activities. Among those who reported that they had a registration book, the presence of the book was confirmed by the interviewers in 73.1% of the cases. Of the registration books that were reviewed by the interviewer, 86.5% had a list of households that the CGPP CV had visited, 92.4% had a list of currently pregnant women, 82.3% had a list of children currently younger than 1 year, and 64.6% had a list of the children who needed immunization. Of the respondents, 74.4% confirmed that they still had the community-based surveillance training manual provided by the CGPP to use as a reference and 66.3% of respondents kept copies of the reports they had submitted. It should be noted that many CVs in remote pastoralist areas do not read or write. Therefore, these CVs submit their reports verbally to HEWs, who then wrote them down and sent them to the next higher level. These documents were not reviewed as part of the study.woreda health office staff. Of the respondents, 30.1% said they had been supervised by a HEW, 21.2% by a staff member at the primary health-care center staff, 19.9% by a CGPP staff member (either a staff member of the secretariat office or of an NGO partner), and 32.0% had not received any supervision in the previous 3 months.Of the respondents, 68.0% reported that they had been supervised in the previous 3 months. Among those who had been supervised, one-half (54.0%) had been supervised by someone working for a CGPP NGO implementing partner and 46.2% by one of the CORE Group Polio Project CVs were asked about what contributions they thought they had made to the Health Extension Program. Almost all (98.2%) stated that their work had brought improvements. As shown in kebele administrators; and more support from primary health-care center staff and woreda health officers.Of the CGPP CVs, 72.3% of reported that they did not face any problems that hindered them from effectively accomplishing their activities. However, virtually all the respondents (94.9% or more) indicated that they would like more support such as more collaboration from community members; more training, information sharing, and supervision support from the HEWs; more collaboration from The CGPP/Ethiopia has been a pioneer in the use of CVs for immunization promotion and surveillance in hard-to-reach and high-risk areas by training and supporting more than 11,000 CVs for supporting the polio eradication program and secondarily for promoting routine immunizations, disease surveillance , health education about pregnancy, delivery and newborn care, promotion of antenatal care at the health post, and promotion of delivery at the primary health-care center.kebele for more than 10 years. Drawing CVs from the community and involving the community in their selection ensures a high level of trust between the CVs and the community. Although CGPP provides training and supportive supervision, 19.3% of CVs reported that they did not receive training before deployment. To ensure the success and capabilities of CVs, additional training is needed.Our findings indicate that of 675 CVs interviewed, 81.1% reported that the community had participated in their selection. A majority of CVs had been living in their CVs are active and well linked to their supervising HEW and to the health system, and they feel that their efforts have been useful for a variety of important aspects of the Health Extension Program. CVs’ knowledge about vaccine-preventable diseases and AFP is reasonably good (94.4%), but only 70.8% and 60.1% knew that measles and tetanus can be prevented by immunization, indicating knowledge gaps. Evidence regarding the knowledge of CVs about immunizations might have been more favorable if the respondents had been prompted on these topics after being given the opportunity for spontaneous responses.Most CVs conduct house-to-house visits to deliver immunization messages to mothers and caretakers, to register pregnant mothers and newborns, to identify those who are behind on their immunization schedule, and to notify the health facility of children needing immunizations.Most CVs report that they are closely linked with their supervising HEWs and others in the health system who supervise them, and most of them report that they attend monthly meetings and submit monthly reports. However, 16% of respondents reported that they do not attend the monthly meeting because of lack of time or the long distance to the meeting venue. The activities of CVs are being documented well, and their activities reach down to every household for which they are responsible. They have a registration book of their activities that includes lists of households, pregnant women, children younger than 1 year, and immunization defaulters. However, illiteracy remains a barrier to documentation in some remote pastoralist areas. Illiterate CVs report orally to their supervising HEW who then write the information on to the monthly report form. Future studies should examine the utility of this process. They also keep the community-based surveillance training manual provided by the CGPP to use as a reference along with copies of the reports they submit to the health facility.5 Although there is a now-emerging, appropriate strong push globally to develop full-time, salaried Community Health Worker (CHW) cadres in low-income settings,14 the contributions of CGPP CVs documented in this article provide strong evidence that community-based volunteers can play an important role in addressing priority health problems, as have other recent reports.20The contributions of CGPP CVs are not limited only to the activities mentioned in this article. They have also played an important role in improving polio immunization coverage in the CGPP catchment areas, as reported in a companion article in this series.Considering the situation of geographically inaccessible and hard-to-reach areas and the nomadic lifestyle of the population, the CGPP CVs have played an important role in promoting polio immunization and other basic health services. They have also contributed to the surveillance of vaccine-preventable diseases  and to increasing the knowledge of mothers about basic maternal and child health. Training, supportive supervision, and regular meetings have all improved the linkage of the CGPP CVs with the Health Extension Program and facilitated their contribution toward eradication of polio and control of other vaccine-preventable diseases. This strong performance of the CGPP CVs reflects a dedicated commitment from partners, including the local, national, and international NGOs who work with the CGPP, the government, and the polio eradication program in Ethiopia. Moving forward, community-based volunteers will be a valuable resource for addressing global, national, and local health priorities in resource-constrained settings.The study also identified some areas where the CGPP CV program can be strengthened. Formal training of all CVs is necessary before field deployment. It is critical that attention be given to the elements that affect CV effectiveness throughout the implementation of the program. Training and supportive supervision are essential to maximize the effectiveness of CVs. The CGPP and other stakeholders need to carefully consider providing timely trainings before deployment, and supportive supervision must receive priority. Continued refresher training will also help build knowledge and capacity that will exist after CGPP is no longer operational. Training should continue to reinforce the understanding of polio vaccination and AFP detection while also increasing knowledge about other vaccine-preventable diseases.


MTOR associated protein, eak-7 homolog (mEAK-7), activates mechanistic target of rapamycin (mTOR) signaling in human cells through an alternative mTOR complex to regulate S6K2 and 4E-BP1. However, the role of mEAK-7 in human cancer has not yet been identified. We demonstrate that mEAK-7 and mTOR signaling are strongly elevated in tumor and metastatic lymph nodes of patients with non-small-cell lung carcinoma compared with those of patients with normal lung or lymph tissue. Cancer stem cells, CD44+/CD90+ cells, yield elevated mEAK-7 and activated mTOR signaling. mEAK-7 is required for clonogenic potential and spheroid formation. mEAK-7 associates with DNA-dependent protein kinase catalytic subunit isoform 1 (DNA-PKcs), and this interaction is increased in response to X-ray irradiation to regulate S6K2 signaling. DNA-PKcs pharmacologic inhibition or genetic knockout reduced S6K2, mEAK-7, and mTOR binding with DNA-PKcs, resulting in loss of S6K2 activity and mTOR signaling. Therefore, mEAK-7 forms an alternative mTOR complex with DNA-PKcs to regulate S6K2 in human cancer cells.  •mEAK-7 forms an alternative mTOR complex with DNA-PK in human cancer•mEAK-7 is upregulated in the metastasized lymph nodes of patients with NSCLC•DNA-PK is essential for mEAK-7-mTOR-S6K2 signaling in human cancer•mEAK-7 is required for cancer cell clonogenicity and spheroid formation Biological Sciences; Cell Biology; Cancer C. elegans (Aberrant mechanistic target of rapamycin (mTOR) signaling has been observed in many types of human cancer . Recentl elegans , the extmEAK-7 uses the S6K2/4E-BP1 axis to regulate mTOR signaling . S6K2 siin vitro and in vivo through RNF168  signaling is activated in response to DNA damage, as indicated by S6 regulation, a crucial readout of mTOR signaling (Cancer stem cells (CSCs) are known to be radiation resistant and thrive under genotoxic stress, but the molecular mechanisms responsible for these adaptations remain unknown . CSCs arignaling . This suGiven that genotoxic stressors are capable of activating mTOR signaling, select CSCs were found to demonstrate radiation resistance, and because CSCs require mTOR signaling, we sought to determine the extent to which mEAK-7 contributes to radiation resistance and self-renewal in cancer cells through an alternative pathway involving mTOR.MEAK7 in many human tissues   and acute myeloid leukemia  had enhanced MEAK7 expression  C. In add (NSCLC) . Therefoigenesis D and 1E igenesis . Also, apression B and S1Cpression . Thus th240/244) p-S6 protein levels were significantly elevated in the primary human tumor when compared with the normal adjacent tissue (240/244) p-S6 protein levels were significantly greater in the metastatic lymph nodes when compared with both primary tumor and normal adjacent tissue (240/244) p-S6 protein levels  p-S6 and a Kaplan-Meier Survival Curve analysis illustrated that mEAK-7 protein levels were strongly associated with poor patient prognosis, specifically in patients with relapse following surgical intervention  for 48 h and 50,000 cells were seeded into invasion chambers for 24 h. Results demonstrated a statistically significant reduction in cell invasion after 24 h  p-S6K1 protein levels (65) p-4E-BP1, an indicator of mTOR signaling mEAK-7. Through hemagglutinin (HA)-mEAK-7 immunoprecipitation (IP) and mass spectrometric (MS) analysis, a list of proteins that potentially interact with mEAK-7 was generated . DNA-depTo confirm our IP/MS results, protein lysates were harvested from H1299 cells stably transduced with HA-mEAK-7. Exogenous mEAK-7 interacted with endogenous DNA-PKcs B and 5C.Because mEAK-7 interacts with mTOR in increasing amounts of amino acid, insulin, or nutrient conditions , we sougFirst, to determine the extent to which S6K2 is required for mTOR signaling following X-ray irradiation, H1975 and H1299 cells were treated with controls or two unique S6K1 or S6K2 siRNAs. Results revealed that p-S6 levels were abrogated after S6K2 knockdown in H1975 and H1299 cells A and S4BAlthough we previously established the function of mEAK-7 in mTOR signaling under nutrient conditions , its rolNext, to determine the relative binding affinities of S6K1 and S6K2 to their most relevant complex, we transfected H1299, H1975, and HEK-293T cells with HA-S6K1 or HA-S6K2 and immunoprecipitated HA-S6K1 or HA-S6K2 to probe for mTOR, raptor, and DNA-PKcs binding. Interestingly, we discovered that HA-S6K1 is capable of binding to DNA-PKcs, but HA-S6K2 binds to DNA-PKcs to a significantly larger extent H. Furthe−/− HEK293T cells  p-S6 levels were measured in response to siRNA-mediated knockdown of mEAK-7, S6K1, or S6K2 and treatment with 10-Gy X-ray irradiation. H1975 cells were treated with control, mEAK-7, S6K1, or S6K2 siRNA for 48 h in DMEM with 10% FBS. Subsequently, the cells were treated with 10-Gy X-ray irradiation for 30 min or 1 h. mEAK-7 and S6K2 knockdown each markedly reduced (Ser240/244) p-S6 levels, but S6K1 knockdown did not have a substantial effect on (Ser240/244) p-S6 levels (389) p-S6K1 levels following X-ray irradiation-induced damage, suggesting a specific role for S6K2 and mEAK-7 during X-ray irradiation-mediated mTOR signaling  p-mTOR levels -mediated mTOR signaling in a dose-dependent manner, but had little effect on (SerR levels A. To detR levels E. These R levels B. In conSurgical intervention and radiation therapy are common treatment modalities for patients with solid tumors. However, in many patients the condition relapses as tumors acquire resistance through intratumoral evolution . CD44+/CAlthough CD44+/CD90+ cells demonstrate radiation resistance and self-renewal capacity that correlate with elevated mEAK-7 protein levels, loss of mEAK-7 alone does not result in enhanced cell apoptosis in human cancer cells . The comAlternative mTOR signaling appears to be upregulated in human patients with cancer, specifically in patients with metastatic disease A–2C. HerIn mini pigs, mTOR signaling is enhanced in salivary glands 5 days after treatment with X-ray irradiation . In addihigh patients have poor prognoses, and that mEAK-7 is essential for self-renewal and radioresistance. To determine the evolutionary benefit that cancer cells gain from upregulating mEAK-7, future research should be focused on elucidating the mechanisms allowing tumorigenesis in a broader range of cancers. Likewise, development of mEAK-7 inhibitors may benefit patients with metastatic cancers that demonstrate aberrant mTOR signaling associated with high levels of mEAK-7.As the role of mEAK-7 is further studied in the context of human disease, its unique role in nutrient-sensing and DNA damage response F will liin vivo. Also, our inhibitor studies against DNA-PKcs, although severalfold more specific to DNA-PKcs versus mTOR, could yield some off-target effects and DNA-PKcs knockout cell lines may not recapitulate all of physiology, suggesting that genetic approaches in vivo are required to understand the role of DNA-PKcs binding to mTOR to form mTORC3. Thus the role of this alternative complex to canonical mTOR signaling requires further study in animal models where mTOR signaling is required for eukaryotic development and disease progression.Here, we report that DNA-PKcs is a binding partner of the mEAK-7-mTOR complex. Some pitfalls of these studies are that we lack animal models that could recapitulate human disease. As most of our work is to identify novel binding partners and the detailed mechanism by which they interact, future studies will be required to examine the role of mEAK-7 All methods can be found in the accompanying


Giardia duodenalis is a zoonotic parasitic protist and poses a threat to human and animal health. This study investigated the occurrence of G. duodenalis infection in post-weaned calves from Sichuan province, China. Faecal samples were collected from a total of 306 post-weaned calves (3–12 months old) from 10 farms, including 4 intensive feeding farms and 6 free-ranging farms. The overall infection rate of G. duodenalis was 41.2% (126/306) based on the PCR results at any of the three genetic loci: beta-giardin (bg), triose-phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. Giardia duodenalis assemblages E , A , and A mixed with E  were identified among the 126 positive specimens. Multilocus sequence typing of G. duodenalis revealed 34 assemblage E multilocus genotypes (MLGs), 1 assemblage A MLG and 7 mixed assemblage (A and E) MLGs. The eBURST data showed a high degree of genetic diversity within assemblage E MLGs. The phylogenetic tree revealed that MLG E3 was the primary MLG subtype in Sichuan province and also the most widely distributed in China. Giardia is one of the most common parasitic protists that infects both humans and animals, poses a considerable threat to human and animal health globally [Giardia, only Giardia duodenalis can infect humans and animals  [Giardia is relatively simple; involving two developmental stages of rapid multiplying trophozoites and infectious cysts, transmitted via the faecal-oral route  [Giardia usually show symptoms such as diarrhoea, abdominal cramps, weight loss, malabsorption or recessive infections without obvious clinical symptoms [globally , 2. Amonanimals) , 4. The or food) , 5. Humasymptoms . Young asymptoms .Giardia duodenalis is recognised as a complex comprised of at least eight different assemblages (A–H) [G. duodenalis assemblage E. Although there are fewer reports of zoonotic assemblages A and B, cattle are recognized as the contributor of the zoonotic sources of infection [G. duodenalis in dairy calves, and the occurrence has been found to be significantly different between pre- and post-weaned stages [G. duodenalis infection in pre-weaned calves in Sichuan province, China [es (A–H) , 7. Assees (A–H) . Cattle nfection . Many red stages –13. In Cd stages , Xinjiand stages , Hubei [d stages  and Guand stages  provincee, China . HoweverG. duodenalis in post-weaned calves from Sichuan province by using multilocus genotype (MLG) data and by assessing the zoonotic potential.In this study, we further investigated the occurrence and genetic diversity of http://ngcc.sbsm.gov.cn/).A total of 306 faecal samples were collected from post-weaned calves (3–12 months old) from 10 farms in 10 regions in Sichuan province, southwestern China, from May to November 2018 . At the Fresh faecal samples  were collected directly from the rectum of the study calves using disposable gloves, transferred into disposable plastic bags, and then stored in 2.5% potassium dichromate at 4°C prior to DNA extraction.This study was reviewed and approved by the Research Ethics Committee and the Animal Ethical Committee of Sichuan Agricultural University (DYY-S20174604). Permission was obtained from the farm owners before collecting the fecal samples.Giardia PCR.Before DNA extraction, stored faeces were washed with distilled water to remove the potassium dichromate. Genomic DNA was extracted from ~250 mg of the individual samples using the PowerSoil DNA isolation kit , according to the manufacturer’s instructions. All DNA samples were stored at −20°C prior to analysis by Giardia duodenalis DNA was detected by nested PCR amplification of the beta-giardin (bg), triose-phosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes. The primers and amplification conditions used in this study have been described previously [eviously . Positivhttp://blast.ncbi.nlm.nih.gov) and Clustal X .All positive secondary PCR products were sent to BGI Tech Solutions (Liuhe Beijing) Co., Limited and were sequenced in both directions. Sequences were aligned with reference sequences from GenBank using BLAST . All the concatenated MLGs were used in a neighbour-joining analysis, with the Kimura-2 parameter model calculated using the Molecular Evolutionary Genetics Analysis (MEGA) version 7 (http://www.megasoftware.net/). The genetic pedigree of the assemblage E MLGs in Sichuan was assessed by using eBURST 3.0 (http://eBURST.mlst.net).Specimens that were successfully subtyped at all three loci were used to investigate the MLGs of G. duodenalis genotypes obtained at bg, tpi and gdh loci in this study were deposited in GenBank under the accession numbers: MK642904-MK642913.The novel G. intestinalis prevalence among the different regions was analysed by χ2 test using SPSS Statistics version 20.0. Differences were considered significant at P < 0.05.The variation in G. duodenalis as a common and widespread pathogen in post-weaned dairy calves in Sichuan province, China. Based on the PCR results at any of the 3 genetic loci , 126 (41.2%) of the 306 faecal specimens tested positive for G. duodenalis, which was similar to the occurrence in Hubei  [G. duodenalis [G. duodenalis. Numerous studies have reported that the prevalence of G. duodenalis is inversely associated with animal age [G. duodenalis in post-weaned calves (41.2%) compared with previous measurements of pre-weaned calves  [P < 0.01, X2 = 76.623, df = 1); which was similar to some reports from China [P = 0.179, X2 = 1.809, df = 1). Similarly, a study conducted in Malaysia also showed no significant differences in the occurrence of G. duodenalis infection between intensive and semi-intensive farms [Infected animals were detected from all of the 10 examined farms, with prevalence ranging from 6.7% to 63.3% . This st, 28/74) , but muc, 28/74) , Liaonin, 28/74) , Heilong, 28/74) , Shaanxi, 28/74) , Xinjian, 28/74)  and Guan, 28/74) . Compare, 28/74) ; Malaysi, 28/74) , but low, 28/74) , 21, 22.imal age . However8, 9.4%)  , assemblage A , and mixed assemblage (A and E)  were identified among the 126 genotyped specimens  that were previously unpublished. Of the tpi subtypes, eight subtypes identified before and the remaining one sequence subtype E25 (MK642913) was previously unpublished. Of the gdh subtypes, ten were known and five were previously unpublished: E21–E25 (MK642908-MK642912). Of these subtypes, the most common were E9 , E3  and E10 . The high genetic diversity of assemblage E in this study was consistent with previous studies [Of the 126 pecimens , which i E n = 11, 91.3%,  studies , 27, 28, studies , 29.bg, tpi and gdh genes was used to systematically characterize intra-assemblage genetic diversity and to determine the genotype of G. duodenalis. A total of 94 specimens were successfully sequenced at the bg, tpi and gdh loci, which formed 34 assemblage E MLGs Click here for additional data file.S2 Figbg, tpi and gdh gene sequences, bootstrap values greater than 50% from 1000 replicates are shown. Isolates from the present study are indicated by black triangles, and isolates from our previous study [The phylogenetic tree was constructed using a concatenated dataset of us study  are indi(TIF)Click here for additional data file.


We report human T-cell lymphotropic virus type 1 infection associated with self-flagellation in 10 UK residents. These persons were heterosexual men from Pakistan, India, and Iraq. One person showed seroconversion in adulthood; 1 was co-infected with hepatitis C virus. No other risk factors for bloodborne virus acquisition were identified. Onward sexual transmission has occurred. Human T-cell lymphotropic virus type 1 (HTLV-1) is transmitted sexually, by contaminated blood products, by organ transplantation, or from mother to child. The estimate of 5–10 million global infections excludes 85% of the general population, for which testing has not occurred, and is probably an underestimate . The patient was of Indian origin and had lived in the United Kingdom since early adulthood. He provided no history of receiving blood products, tattoos, or injection drug use. No family history was suggestive of HTLV-1 infection. In 2008, he donated blood in the United Kingdom that was tested for HTLV-1; he was seronegative. His wife of 10 years was also negative for HTLV-1. He had engaged in zanjeer voluntarily during childhood outside the United Kingdom and continued this practice. In the United Kingdom, the blades were soaked in a bucket containing an over-the-counter antiseptic solution, along with the blades of other men conducting the practice simultaneously. In the previous few years, his practice had also involved striking his forehead with a knife, which was subsequently shared by other men. Physical examination showed widespread scarring on his back  and the We provide epidemiologic findings and HTLV-1 proviral load  for this case-patient and 9 other asymptomatic HTLV-1 carriers of similar demography who reported a history of self-flagellation in Iraq, Pakistan, India, or the United Kingdom . Most reAll 10 patients were given a diagnosis of infection with HTLV-1 through screening programs since 2013. Nine patients had strongly positive Western blot results and positive PCR results. One patient had indeterminate Western blot results and negative PCR results but an HTLV-1/2 enzyme immunoassay sample/cutoff ratio >80, which is consistent with HTLV infection  do not ask about the practice and do not screen for HTLV. We propose that self-flagellation be added to the list of risk factors that result in testing for bloodborne viruses, including HTLV-1. Blood transfusion services might screen for this practice when assessing potential blood donors; for 6 men in this study, blood donation was the route of diagnosis. However, screening blood donors for HTLV-1 infection is not universal. Absence of screening, particularly in regions where self-flagellation is practiced, could accelerate dissemination of this infection. The seroprevalence of HTLV in Pakistan is unknown, but a recent study reported a prevalence of 0.19% among low-risk blood donors , which places them at risk for HTLV-associated disease. In all but 1 case-patient, HTLV-1 was the only bloodborne virus detected. Within heterosexual populations, transmission of HCV is commonly associated with using contaminated injection equipment. HTLV-1 predominance in our cohort suggests the presence of a pool of monoinfected persons and spread within this international community.All patients have been advised by medical practitioners not to share implements during self-flagellation and to encourage fellow practitioners of flagellation to be tested for bloodborne viruses. Our visits to communities in which these practices occur to discuss risk elimination, raise awareness, and promote testing facilities have been favorably received, and risk reduction has been implemented.


Signaling activation is a tightly regulated process involving myriad posttranslational modifications such as phosphorylation/dephosphorylation, ubiquitylation/deubiquitylation, proteolytical cleavage events as well as translocation of proteins to new compartments within the cell. In addition to each of these events potentially regulating individual proteins, the assembly of very large supramolecular complexes has emerged as a common theme in signal transduction and is now known to regulate many signaling events. This is particularly evident in pathways regulating both inflammation and cell death/survival. Regulation of the assembly and silencing of these complexes plays important roles in immune signaling and inflammation and the fate of cells to either die or survive. Here we will give a summary of some of the better studied supramolecular complexes involved in inflammation and cell death, particularly with a focus on diseases caused by their autoactivation and the role autophagy either plays or may be playing in their regulation. In order to assemble supramolecular signaling complexes in a tightly regulated fashion, certain protein–protein interaction domains and motifs have evolved. By using shared interaction mechanisms, these structures can assemble many subunits of differing function in a rapid and modular fashion to facilitate signal transduction. There are likely many other examples of proteins and domains that fall into this category, but in this review we have chosen to focus on the following structural elements due to their significant representation in the pathways regulating cell death and inflammation.The death domain containing protein family is involved in numerous aspects of cell signaling and fate and contains several subfamilies including Caspase Activation and Recruitment Domain (CARD), Death Effector Domain (DED), Pyrin Domain (PYD), and the Death domain (DD) itself . These dAnother structural element contained in supramolecular complexes discussed in this review and which has been shown to play a crucial role in inflammatory and cell death signaling is the Receptor Homotypic Interaction Motif (RHIM) . RHIMs aA common component and key player in the regulation of supramolecular signaling complexes is ubiquitin. In all complexes described in this review, polyubiquitin chains are attached to one or more of the subunits. A common signal activating function of ubiquitin chains in these complexes is to recruit kinase complexes IKK and TAB/TAK to activate NF-κB. However, the same K63 chains are also involved in the eventual silencing of the signaling complexes (see later). Other linkage specific chains are also present including K48 and Met1. Met1 which is added by the Linear UBiquitin chain Assembly Complex (LUBAC) is also important for NF-κB activation through recruitment of IKK complexes in a similar fashion to and in cooperation with K63 chains . K48 chaAssembly for supramolecular complexes such as those discussed throughout this review, presents a potential problem in terms of switching the signal off. Given the damaging outcome of overactivation of inflammatory or cell death promoting complexes, these structures need to be silenced before they can lead to cellular or tissue damage and disease. While this may in part be regulated by post translational modifications such as (de)ubiquitination, and (de)phosphorylation the likely stability and energetic requirement to break apart complexes such as RHIM or CARD mediated fibrils make this an unlikely mechanism to completely explain disassembly and inactivation of the fully assembled complexes. Degradation of subunits of these complexes by the proteasome may also play some role, however, the size of the complexes discussed in this review make it unlikely that proteasomal degradation of the assembled complexes occurs. Access to the proteasome typically requires unfolding of the substrate to allow it to fit within the catalytic barrel . IndividAutophagy is at its heart a cellular recycling mechanism to provide energy in times of nutrient stress, but has been adapted in multicellular organisms to also regulate multiple aspects of cellular biology. The canonical pathway also known as Macroautophagy creates so-called autophagosomes, membranous vesicles, which engulf random parts of the cytosol and organelles  Figure . These aMuch of the best studied signaling complexes are in the TNF Super Family (TNFSFR), including TNFR1, FAS, TRAIL among others. Each of these receptors ultimately leads to activation of caspase-8 as well as activating transcriptional programs, particularly NF-κB. TNFSFR use death domain family interactions to recruit both scaffolding proteins such as TRADD and FADD, as well as effector proteins such as Caspase-8/10, and RIPK1, although many of the effector proteins also exhibit some scaffolding function, independent of their catalytic activities. Using TNFR1 as a well-studied example, TNFR1 recruits the adapter TRADD, which in turn recruits both RIPK1 and/or FADD. Recruitment of TRAF2/5 along with cIAP1/2 triggers k63 linked ubiquitylation of RIPK1 as well as other components. These ubiquitin chains recruit IKK as well as the TAB/TAK complexes and the linear ubiquitin chain assembly complex (LUBAC) which further adds linear ubiquitin chains. The IKK and TAB/TAK complexes then both activate NF-κB as well as phosphorylate RIPK1 to prevent its activation . This reIn the case of TNF family receptors, one important aspect of their activity is their endocytosis. Endocytosis is required for apoptosis from TNFR1 for example . EndocytToll Like receptors, like TNFSFR, also signal through large complexes which may promote both inflammation and cell death. TLR’s, use specific adapter proteins to recruit signaling complexes to the receptor. More specifically, TLRs that signal through the adaptor TRIF/TICAM1 are able to recruit RIPK1 and through RIPK1 activate caspase-8 and or RIPK3 to trigger apoptosis or necroptosis  Figure , 4 TRIF In either case, both MYDD88 and TRIF adapters have been shown to form supramolecular complexes which are required for several aspects of their signaling activity . Indeed A number of studies have now associated regulation of TLR signaling via autophagy. Specifically Myd88 was reported to be recruited to p62 and Histone deacetylase 6 (HDAC6) positive structures upon TLR4 stimulation . p62 actTRIF can form large fibrillar complexes that have been variably reported to interact with the autophagy cargo receptors p62, Tax1BP1, and NDP52 to target them for degradation . Our ownAs with TNFRSF receptors that become endocytosed, TLRs, in part at least, are associated with endosomes, an aspect which is essential for their activity . In addiInflammasomes are another example of a death domain based supramolecular complex that has potent inflammatory signaling activity but can also trigger death of cells. There are a number of inflammasomes, however, the majority share a core structure, with a receptor subunit such as NLRP3 activating and recruiting Apoptosis-associated speck-like protein containing a CARD (ASC), which is able to then polymerize into a helical fibrillar structure that recruits Caspase-1 . This brSTING is activated by cyclic dinucleotides (cGAMP) produced by cGAS upon detection of cytoplasmic DNA . cGAMP bSTING activation can also induce apoptosis in T cells and B-cell lymphomas although the mechanism is still unclear . Auto acSTING has also recently been identified as an autophagic substrate. Upon activation it binds to p62 and is targeted for degradation through autophagy . STING iMitochondrial antiviral-signaling protein (MAVS) is a signaling scaffold for detection of viral RNA products. The receptors RIG-I and MDA5 bind to viral dsRNA molecules and then oligomerize . ExposurWhile MAVS appears to be an autophagy substrate it is perhaps a somewhat special case in this regard due to its location on the mitochondria. MAVS is reported to contain an LIR motif for direct recruitment of LC3 and MAVS activation and assembly into its large fibrillar state can stimulate mitophagy , thus reNOD2 is another CARD containing PRR that recognizes bacterial muramyl dipeptide (MDP) . ThroughNOD2 has a complex interaction with the autophagy machinery. As is the case with most of the complexes discussed here, NOD2 can induce autophagy. It is thought that this occurs through a RIPK2 kinase dependent mechanism . AdditioIn a similar manner to inflammasomes, the CARMA-BCL10-MALT1 (CBM) signaling complex consists of a core of BCL10 that recruits the paracaspase MALT-1. Different CARD containing adapters can recruit BCL10 to trigger its assembly including CARD9, -10, -11, and CARD14 . CARD11/Interestingly, mutations in CARD11 (Carma1) are causative of B cell expansion with NF-κB and T cell anergy (BENTA), an immunodeficiency caused by overactivation of the CBM complex. These mutations cause CARD11 to aggregate and recruit BCL10 and MALT1 into large complexes . They arAs with the other supramolecular complexes discussed so far, a link with autophagy has been shown with CBM signalosomes too. Degradation of BCL10 in response to TCR stimulation occurs through a proteasome independent lysosomal dependent mechanism , suggestMitochondria act not only as the metabolic engine of the cell, but also as a key signaling hub filtering signals for cellular growth and energetics, innate immune and inflammatory signals, and also decisions to survive or die. The classical intrinsic mitochondrial apoptotic machinery comprised of Bax and Bak mediated pores regulated by BCL2 family pro and anti-apoptotic proteins is well characterized, if not completely understood . As withThe central role mitochondria play in regulating so many aspects of cellular homeostasis and immunity mean that they can in a sense be considered as a supramolecular signaling hub themselves. Given their importance it is not surprising then, that they are also heavily regulated and prone to quality control. Mitophagy is a specialized form of autophagy that causes the degradation of damaged mitochondria . MitophaAn interesting connection between the activation of these receptor signaling complexes and their degradation by autophagy is the activation of the Tank Binding Kinase 1 (TBK1). TBK1 is required for IRF activation from many of the receptors complexes described in this review including TLR3/4 via TRIF, STING and MAVS. TBK1 is also a well characterized activator of autophagic cargo receptors including p62, optineurin and NDP52 . TBK1 phIn another twist to the role of autophagy in regulating supramolecular signaling complexes, some cargo receptors such as p62 can promote signaling and assembly of the complexes prior to their degradation . This haListeria monocytogenes (Recently a role for HDAC6 has been shown in clearance of ytogenes . In thisytogenes . In a siytogenes , 2014, hytogenes . NOD2 alytogenes . Of noteytogenes . Additioytogenes . TogetheA number of diseases are associated with deficiencies in autophagy, many of which are inflammatory in nature and in a number of cases show direct links to proteins from supramolecular signaling complexes involved in cell death and inflammatory signaling. Gaucher’s Disease is a lipid storage disease caused by mutations in glucocerebrosidase that results in accumulation of the sphingolipid glucocerebroside in lysosomes, effectively blocking their function. Thus, as a byproduct, the autophagy pathway is also backed-up and blocked by a failure to degrade targets in the lysosome . GaucherNiemann Pick disease is another lysosomal disease that is associated with inflammatory pathology, particularly Crohn’s disease like symptoms. Niemann Pick diseases are caused by failure to metabolize Sphingomyelin for various reasons, leading to lysosomal disfunction . While iFormation of large supramolecular complexes as signaling hubs is emerging as a unifying theme in signaling and it is likely that many more receptor complexes will demonstrate this capacity. The fact that so many receptors all share common structural domains and signaling targets supports this and highlights the crosstalk that many of these receptors have in regulating the strength, and severity of signals that are produced. The propensity of these complexes to assemble is also highlighted by the numerous, although rare, genetic immune diseases associated with auto-activation of components of the complexes discussed here. The common theme of induction of autophagy, recruitment to cargo receptors to amplify signals prior to their degradation provides a tightly regulatable circuit for negative regulation of these important signaling complexes and suggest that autophagy induction may provide a useful target in helping to prevent some of these diseases at their source.IG conceived of and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.