We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function.Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed  in-vivo approach using dual-colour reporter transgenesis in zebrafish for unambiguous assessment of the effects of disease-associated CRE mutations on CRE activity during the entire time-course of embryonic development. The highly efficient, cost-effective and modular design of the assay allows rapid analysis of several CRE-variants in parallel. We illustrate the robustness of our approach using examples of CRE-variants associated with a broad spectrum of genetic diseases including brain, limb, eye and jaw disorders. In a single assay the method can address where and when in development the CRE variant affects its activity, what potential target genes are misregulated by the change and what upstream trans-acting factors are likely to mediate this effect.Cis-regulatory elements (CREs) play a vital role in gene regulation by providing spatial and temporal specificity to the expression of their target genes. Understanding how these regions of the genome work is of vital importance for human health as it has been demonstrated that genetic changes in these regions can result in incorrect gene expression, leading to a variety of human diseases. Functional characterization of putative CREs and the effects of mutations on their activity is currently a major bottleneck in many studies towards understanding the causes and mechanisms of disease and disease susceptibility. We describe a robust  The lack of efficient high-throughput and physiologically relevant assays for functional validation of tissue- and developmental-stage-specific CREs and identification of their target genes thus poses a real bottleneck in translating human non-coding variation into better understanding of disease mechanisms.Cis-regulatory elements (CREs) such as enhancers are vital functional regions of the genome which determine where, when and at what levels their target genes will be expressed . In keepDanio rerio). Zebrafish embryos are transparent and develop rapidly and outside the mother, making it feasible to visualize specific tagged cell types in the living animal. They are well suited for transgenic manipulation since it is relatively easy to collect and inject large numbers of fertilized eggs [In our search for an efficient approach for characterizing regulatory variation in a vertebrate model system, we developed a versatile, streamlined assay pipeline using zebrafish  [To determine the performance of the system, we tested a set of known disease-associated CREs in dual colour zebrafish reporter transgenics and demonstrate that the method is effective in revealing differences in functional output between variants in agreement with previously reported in mouse studies of these elements. We then applied the method to investigate a set of novel putative CREs with potential disease-associated variants in the genomic region of the ce (PRS) . ConsistConnecting a sequence variant in a non-coding genomic region to disease aetiology in the patient in whom the variant was detected, is currently a major bottleneck in human genetics.in vivo comparisons of the spatial and temporal activities of wild-type (Wt) and putative SNP or mutation (Mut) bearing CREs within the same animal by confocal imaging  and the human (or other species) gene the CRE is thought to regulate.Both Tol2 constructs (bearing the Wt and Mut versions of the CRE) were co-injected with Tol2 mRNA into 1–2 cell stage zebrafish embryos . Since mThe most common established mechanism by which disease-associated sequence changes affect CRE function is by alteration or disruption of binding sites for tissue-specific transcription factors. The final part of our assay pipeline contains a convenient method for testing the validity of predicted disruptions of Transcription Factor Binding Sites (TFBS) by the disease-associated point-mutations . F1 fishTo test the robustness of our dual-fluorescence reporter transgenic assays we selected four disease-associated CREs from the literature for which point-mutations had been firmly established as the cause of human monogenic disease .SHH (sonic hedgehog) expression in the developing forebrain —controls expression that is restricted to the posterior mesenchyme of the distal limb bud  . The minid plate . Our assSOX9, in the interval between SOX9 and KCNJ2  allele of this element at 48 hpf . HoweverWe also observed temporal  changes in tissue-specific CRE activity due to the presence of PRS-associated variants in another of the CREs . P300-PKSOX9, and altered spatiotemporal expression for three of these elements when carrying a sequence variant seen in patients with Pierre Robin Sequence.The final two CREs also revealed distinct craniofacial expression patterns in reporter transgenic embryos. However, for these elements we observed no differences in CRE activities between the wild-type and variant alleles Fig  and 6C. in vivo assays, such as hydrodynamic tail-vein injection and retina explant assays [in vivo assays that enable the unambiguous comparison of putative mutant and wild-type alleles of CREs for spatial and temporal differences in activity. Such screens are vital for differentiating between common, background sequence variation and functionally active, disease-associated mutations in CREs, and will enable rapid prioritization of candidates for functional follow-up studies.The importance of intact gene regulation for correct development and homeostasis has become increasingly recognised. As a result, research groups interested in understanding the aetiology and causative mechanisms of human disorders or disease susceptibility are now routinely looking for possible mutations or variations in CREs in patient cohorts, especially in cases where convincing causative mutations could not be found in the coding regions of genes. However, the lack of efficient and physiologically relevant assays to functionally validate the consequence of these sequence changes and to distinguish disease-linked changes from background sequence variation is hampering the translation of knowledge about human genetic variation into an understanding of disease mechanisms. CRE activity is highly dependent on the availability of the right combination and stoichiometry of specific transcription factors. Consequently, high-throughput assays for assessing the potential effects of disease-associated SNPs in cultured cells are largely uninformative as they often lack the relevant and necessary biological context for tissue or cell-type specific CRE function. In future, cell lines reflecting more closely the cell phenotypes of developing tissues may become available through emerging stem cell differentiation protocols . A few ht assays –23 have The novel dual-colour zebrafish reporter transgenic assay pipeline that we have developed for investigating the functional consequences of disease- and disease-risk associated CRE mutations is designed to incorporate many of the above described merits, and thus will be a valuable tool for analysing effects of CRE mutations identified in patient cohorts and GWAS studies. While we cannot control for the number and position of transgene insertion sites in our assay, multiple independent transgenic lines bearing both the Wt and Mut version of the element driving distinct fluorescent reporters in the same animal can rapidly be constructed and tested, creating a clear consensus activity pattern and minimising the chance of bias in the analysis introduced by position effects due to site of integration of the transgenes. Other methods aimed towards targeted integration of transgenes in pre-defined docking sites in the zebrafish –27 or meCharacterisation of detailed expression patterns driven by particular elements allows selection of the most likely candidate sequences by virtue of their expression in the relevant anatomical structures. In combination with information on the expression patterns of the genes located within the relevant genomic region this can also help to find the likely target gene of an enhancer. This can otherwise be problematic as target genes of enhancers are often not simply the nearest one in the genome, but can be found hundreds or even thousands of kb away in either direction, beyond intervening non-target genes, and may reside within the introns of other non-target genes , 30. MosDisruption of cis-regulatory control of gene expression can occur via a number of distinct mechanisms . These rThe second mechanism we identify is a partial loss of activity in the temporal dimension, as exemplified by the p300-pk17 element in patient PRS026 which loses expression in the oral cavity only at later developmental stage. This indicates that the element functions normally when it first becomes activated, but fails to maintain its activity subsequently. The third example is the complete loss of functional activity of the element in our assay. This suggests the mutation disrupts the binding of an essential factor potentially affecting the effective formation of a TF complex, or enhanceosome  at the ecis-regulatory sequences themselves, while the transcriptional machinery involved in the reading of the elements remains largely conserved due to selection against the wide-ranging effects of changes in the pleiotropic functions of transcription factors. Our results indicate that human regulatory elements implicated in disease can be tested in zebrafish with confidence.Even though not guaranteed to work for all enhancers, our results show that the dual reporter transgenic zebrafish assay is a suitable model system to test enhancer activity of many human disease-associated elements even when the element is not obviously conserved in zebrafish itself. Evolution is characterised by variable loss and gain of cis-regulatory sequences . TeleostSHH and IRF6, for which enhancers were tested in this report.It is remarkable that single nucleotide changes at such large genomic distance from their target gene can have such profound phenotypic effects. Most of the currently known examples of distal cis-elements involved in genetic disease act as enhancers for genes sensitive to haplo-insufficiency . Our demSeveral of the enhancer variants described here are either inherited or even occur as rare SNPs in the wider population. Nevertheless we present strong evidence that these variants affect the function of the regulatory element in which they occur. Mutations in enhancers may not always lead to clear, fully penetrant phenotypes, but rather could contribute partly to a phenotype in specific individuals, depending on the presence of other modifier alleles. Incomplete penetrance may be observed for a number of reasons including variation in expressivity from the enhancer, or redundancy or buffering among multiple enhancers.in vivo animal model system. We anticipate that our strategy will facilitate the analysis of non-coding variants in genome-wide association study (GWAS) hits, and can also be used in studying the mechanistic basis for enhancer activity, the engineering of enhancers with desired properties, or to drive highly spatio-temporally specific expression of genetic tools.The mufti-faceted assay we have described here allows for the functional characterisation of disease-associated enhancer variants at the highest throughput that can currently be achieved using an All zebrafish experiments were approved by the University of Edinburgh ethical committee and performed under UK Home Office license number PIL 60/12763; PPL 60/4418.CREs selected for analysis in transgenic reporter assays were cloned by PCR amplification of the fragment containing it plus flanking sequence from genomic DNA, using Phusion high fidelity polymerase (NEB). attB4 and attB1r sequences were included in the PCR primers for use with the Gateway recombination cloning system (Invitrogen). The amplified fragment was first cloned into the Gateway pP4P1r entry vector and sequenced using M13 forward and reverse primers for verification. In cases where a point mutation was engineered in the wildtype CRE, a site-directed mutagenesis kit  was used on the pP4P1r vector containing the wildtype CRE sequence. Test elements in the pP4P1r vector were combined with a pDONR221 construct containing either a gata2 promoter-eGFP-polyA or a gata2 promoter mCherry- polyA cassette, and recombined into a destination vector with a Gateway R4-R2 cassette flanked by Tol2 recombination sites , 37. ThePurification of genomic DNA from blood lymphocytes and Sanger sequencing of non-coding elements upstream of SOX9 was performed according to standard protocols, using the primers listed in Sequence conservation of disease associated CREs was assessed by multiple sequence alignment and visualised using the VISTA or ECR Browser with the human hg19 sequence as base genome , 39.Zebrafish were maintained in a recirculating water system according to standard protocol . EmbryosEmbryos for imaging were treated with 0.003% PTU (1-phenyl2-thio-urea) from 24 hpf to prevent pigmentation. Embryos selected for imaging were anaesthetized with tricaine and mounted in 1% low-melting agarose. Images were taken on a Nikon A1R confocal microscope and processed using A1R analysis software.A zebrafish Six3 antisense morpholino oligonucleotide (Six3AMO) was obtained from Gene Tools, LLC, with the following sequence: 5’ GCTCTAAAGGAGACCTGAAAACCAT 3’. This morpholino has sequence complementary to the highly conserved sequences around the translation initiation codon of both six3a and six3b, and hence inhibits the function of both zebrafish six3 genes . As contin situ hybridization on fish embryos was performed as previously described [RNA escribed . The seqNuclear extract was prepared from ~100 morpholino injected embryos at 48hpf using NE-PER Nuclear and Cytoplasmic Extraction Reagents . The extracts were boiled with 30 μL of loading buffer  for 5min and were resolved by 10% SDS-PAGE, transferred to nitrocellulose, incubated with antibody (anti-Six3 antibody ab139317), and detected by chemiluminescence . The membrane was then re-probed with anti-α-tubulin antibody (ab44928) as loading control.S1 FigDual reporter fluorescence in transgenic F1 embryos from multiple additional independent lines for the SHH-SBE2 (A), SHH-ZRS (B), PAX6-SIMO (C), p300-Pk19 (D) and p300-Pk17 (E) elements. These lines are independent from the ones shown in the main text. The expression sites that are consistent between the multiple transgenic lines for each of the CREs are marked. RH: rostral hypothalamus; CH: caudal hypothalamus; PF: pectoral fin; L: lens; CMZ: ciliary margin zone; OV: otic vesicle; PA2-5: pharyngeal arch 2–5; OC: oral cavity; PQ: palatoquadrate; MC: Meckel’s cartilage; Wt: wild-type; Mut: mutant; hpf: hours post fertilization.(TIF)Click here for additional data file.S2 FigThe efficiency of Six3 depletion in zebrafish embryos injected with Six3 AMO morpholinos is demonstrated by Western blotting of protein extract from pooled morpholino-injected embryos with anti-Six3 antibody. The absence of Six3 in the extract from Six3 AMO morpholino-injected embryos is highlighted by a black arrow at 32 KDa, while Six3 is present in fish injected with a control morpholino. After stripping, the blot was incubated with anti-α-tubulin antibody for loading control .(TIF)Click here for additional data file.S3 Fig(A) The Wt(G) allele is shown driving eGFP expression in a restricted region of the developing pectoral fin (PF) in two independent founders (1 and 2). (B) The Wt(G) allele is shown driving mCherry expression in a restricted region of the developing pectoral fin (PF) in four independent founders (1–4). Consistent reporter gene expression was detected only in the pectoral fin in all independent founders shown.SHH-ZRS enhancer-driven reporter expression is shown at 72 hpf (TIF)Click here for additional data file.S4 FigGenotypes are indicated for those individuals available for sequencing: + wildtype allele,—mutant allele. Probands are indicated with an arrow.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Knowledge about the spatiotemporal tree growth variability and its associations with climate provides key insights into forest dynamics under future scenarios of climate change. We synthesized 17 tree-ring width chronologies from four tree species at the high-elevation sites in the southeast Tibetan Plateau (SETP) to study the regional tree growth variability and climate-growth relationships. Despite of diverse habitats and different physiological characteristics of these species, these tree-ring chronologies shared a significant common variance in SETP. An unprecedented increase in the shared variance is found along the latter half of the 20th century, coinciding with the enhancement of the frequency of extreme rings among chronologies. It is found that minimum winter temperature tends to be the dominant climate for trees in this region. The site-specific responses in cold (1965–1980) and warm (1990–2005) intervals by means of Fuzzy Cmeans (FCM) clustering reveal that the remarkable enhancement of growth synchrony among trees mainly occur in warm conditions. This is different from previous findings indicating that increased consistence among temperature sensitive tree rings in cold periods. This may be related to the reduced temperature sensitivity of regional tree growth as winter minimum temperature is lower than a certain threshold, which is in agreement with the “principle of ecological amplitude”. In addition, it is worth noting that precipitation in June have started to restrain the tree growth since the beginning of the 1980s, which is possibly an important contributor for synchronized growth among trees in SETP. Climate change exerts one of the major abiotic factors shaping the growth of the terrestrial forests via directly modulating the metabolism of the individual trees and indirectly changing the forest structures , which cThe southeast Tibetan Plateau (SETP), together with its vicinity, is one of the most studied areas in China with a network of relatively densely distributed tree-ring chronologies developed from endemic tree species. Complex climate-growth relationships in the SETP are largely due to its large climate gradients and complex topographic features. High altitude forests in SETP are sensitive to climate variability due to harsh environment conditions that inhabit tree establishment, growth and survival. Temperature-stress appears to be one limiting factor for some coniferous forests in SETP , 14, 15.Increasing evidence has indicated that climate-growth associations are modified by a changing climate in recent decades over a variety of regions. For example, decreased sensitivity of tree growth to temperature since the 1980s, e.g. the so-called “divergence” problem, has been frequently reported from the circumpolar area in the Northern Hemisphere , 22, 23.In this context, we hypothesize that warming and climatic variability have produced changes in tree-growth patterns, as well as in the response of alpine forests across SETP. To test this assumption, a network of pine ring-width chronologies along SETP is collected. The aims of this work are, 1) to detect temporal variability of radical growth and the possible climate determinants; 2) to investigate site-specific response to cold and warm conditions; 3) to detect growth-climate associations and their stability across time.http://www.ngdc.noaa.gov/paleo/ftp-treering.html). The tree-ring samples were collected from four tree species in the climate stressed sites, mainly at open forests growing on shallow soil. The dominant one is Abies forestii Rogers (8 chronologies), followed by Juniperus tibetica Kom. (4), Tsuga dumosa Eichler (3) and Picea likiangensis (Franchet) Pritzel (2). The mean segment length from chronologies of A. forestii is the longest (276.5 yr), and the average chronology length of P. likiangensis is the shortest (243.2 yr).Most of SETP is dominated by a highland continent climate with long, dry winter and cool, wet summer and a large diurnal range of temperature. We synthesized a tree-ring network of 17 chronologies over SETP ; S1 DataBiological trends were removed from the raw data by fitting straight lines or negative exponential curves to retain as much low-frequency variability as possible. Tree-ring series that could not be well fitted by the two conservative curves were detrended by a relatively stiff cubic smoothing spine curve with a 50% cutoff at around 67% of the mean segment length . Tree-riIn total, 19 gridded climate records around the sampling sites  analysis based on the correlation matrix was calculated for the overlapping period 1825–2005 to evaluate the shared variance of the chronology network. Then the first PC (PC1) with all chronologies was computed for the successive periods of 50 years lagged by 10 years in order to evaluate the temporal changes of the shared variability. For evaluation of the tree-growth variability, years with extreme wide and narrow rings (mean ± 1.5SD) were identified for each chronology.Correlation function analysis is employed to quantify the climate-growth relationships between the regional chronology (PC1) and climate variables over a “dendroclimatic year” from the start of the previous growing season (herein May) to the end of the current growing season (September) during the period 1951–2005. Moving correlation functions (MCFs) were further used to investigate the possibly changing relationships between regional tree growth and climate data by adopting a fixed sliding window of 30 years.it is generally impossible to come out the exclusive partitions in ecology due to the unquantifiable disturbances and the complex interactions among ecological patterns or processes [To better investigate the responses of tree growth to relatively warm and cold winters  positive temperature-growth associations are observed in November (0.386), December (0.464), January (0.486) during the pre-growing season. The highest correlation coefficient (0.596) is observed with November-January average minimum temperature.No significant correlation is found between the regional tree growth (PC1) and monthly precipitation during the period from 1951 to 2005 . The corA. forestii woods at ab01, ab03, ab05, ab08 sites respond in a similar way with J. tibetica woods in ju01, ju03, ju04 sites and P. likiangensis woods in pi01, while trees in ab06, ab07, ab08 sites react in a similar way with T. dumosa trees in ju02, ts01, ts02 sites and P. likiangensis trees in pi02 , which means that trees at these sites show no significant sensitivity to climate change.Considering the temperature-growth relationships in SETP, the results of FCM clustering are computed with the indexed tree-ring widths for all the tree-ring sites under cold and warm winter temperature conditions, respectively . As ment in pi02 . Under t02, ts03 . In addiThe MCFs reveal that the climate-growth interactions in SETP have varied through time . It is nThe ring-width chronologies in this study were derived from a wide range of forests of four different species in high-elevation sites across SETP. Despite of this, the PC1 held a significant percentage of common variance for these tree-ring chronologies , suggestTemperature-stress in the pre-growing season appears to be the prevailing climate limitation for tree growth at these sites in SETP as inferred from positive correlations with winter minimum temperature . Warm wiThe increase in consistent growth pattern among trees often occurs when the stress from climate is enhanced , 40, 41.Low sensitivity to winter temperature for tree growth was observed during the cold 1960s-1970s period , indicatIn addition, it is not negligible that regional tree growth has elevated its sensitivity to precipitation in June since approximately the early 1980s , indicatWe have developed 17 tree-ring width chronologies across SETP from four endemic tree species. Despite of the diversity of species and habitats, these chronologies shared a significant common variance (PC1) that can express the large-scale climatic signal in the study area. An unprecedented increase in the shared variance is detected during the latter half of the 20th century. Regional tree growth is mainly limited by winter temperature during the pre-growing season. However, consistence among tree-growth and the temperature-growth relationships become strong (weak) in warm (cold) periods based on the FCM clustering analysis. This is different from previous findings that indicated increased (decreased) consistence among temperature sensitive tree rings in cold (warm) periods. This suggests the presence of nonlinear climate-growth relationships. It is found that tree growths are less sensitive to minimum temperature in freezing winters. Additionally, June precipitation shows significant correlations with tree growth since the beginning of the 1980s, which is possibly related to elevated evaporative demands and degradation of the seasonal frozen soil during the growing season due to the recent warming. Both recent warming in winters and spring drought may induce the coherent growth pattern among trees over SETP.S1 Data(ZIP)Click here for additional data file.S1 FigCorrelations between the regional chronology PC1 and monthly a) mean temperature (green), b) maximum temperature (red) and c) minimum temperature (blue) from previous May to current September during the period 1951–2005. The 95% significance level is indicated by dash lines.(TIF)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


Many plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to a few viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes. Positive-strand RNA [(+)RNA] plant viruses are the most abundant in plant viruses, and cause diverse diseases in host plants. Because of the RNA nature of their genomes, it is not surprising that RNA-binding proteins (RBPs) of viral and host origin affect multiple steps of virus infection. It is known that viral RNA (vRNA) has critical non-template functions in addition to the essential function as a replication template . After eFollowing the translation and replication processes, (+)RNA plant viruses spread their infection into neighboring uninfected cells through plasmodesmata (PD) by using their encoded movement protein (MP). Some plant viruses move through PD in the form of virus particles and others are thought to move in ribonucleoprotein (RNP) complexes that contain MP . MPs froIn this review, we overview the currently defined roles of viral- and host-derived RBPs in virus infection by focusing on the proteins whose RNA-binding activity has been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes. We recommend readers consult several current reviews that discuss other topics relating to the roles of host RBPs during RNA virus infection .Red clover necrotic mosaic virus (RCNMV) has a bipartite genome, RNA1 and RNA2  in its 3′-untranslated region  affinity and immunoprecipitation assay showed that the YRE is necessary and sufficient to interact with p27 and with the 480 kDa replication complex . Interestingly, the p27–YRE interaction is modulated by Hsp90  and related Cucumber necrosis virus (CNV) takes place at the peroxisomal membrane  takes place in ER membrane-associated invaginations or spherules induced by replication protein 1a (in vivo stabilization of RNA2 and RNA3 (RNA replication of otein 1a . In thesotein 1a . Althougotein 1a . The intand RNA3 .Tobacco mosaic virus (TMV) binds the 5′-UTR of the genomic RNA cotranslationally, and that this binding plays an important role in regulation of vRNA translation and replication.A recent publication by cis-acting RNA element essential for TBSV replicase complex assembly and (–)RNA synthesis (in vitro (Eukaryotic translation elongation factor 1A (eEF1A) is an abundant cellular protein that functions in protein degradation, apoptosis, nucleocytoplasmic trafficking, heat shock, and organization of the actin cytoskeleton in addition to its canonical role in delivering aminoacyl-tRNA to the elongating ribosome . Severalynthesis . Pharmacin vitro . These fin vitro .Bamboo mosaic virus (BaMV) RNA has been detected in chloroplasts, a putative replication site of BaMV (Nicotiana benthamiana can bind to the 3′-UTR of BaMV (+) RNA  is inhibited, suggesting that PGK is required for the replication of BaMV. Interestingly, overexpression of cytosolic- or nuclear-localized PGK mutants has detrimental effects on BaMV infection. This is consistent with the author’s prediction that these PGK mutants mislead BaMV RNA away from chloroplasts to the cytoplasm or the nucleus that are unsuitable sites for viral replication. BaMV seems to use the trafficking system of chl-PGK to target vRNA to the chloroplasts for replication. In support of this, artificial chloroplast-targeting EF1a, which binds to BaMV (+)RNA, can partially complement BaMV accumulation in chl-PGK-knockdown N. benthamiana ( of BaMV . Chlorop (+) RNA . PGK is  (+) RNA . The rol (+) RNA . In protthamiana . BaMV RNthamiana . It willCucumber mosaic virus (CMV), and that BRP1 plays an important role in the nuclear importation and multiplication of the satellite RNA. BRP1 also binds Potato spindle tuber viroid RNA both in vitro and in vivo, and is involved in the nuclear transportation and multiplication of the viroid  forms a high concentration salt-resistant vRNP (Turnip crinkle virus (TCV) MP is a restricting factor for systemic infection by the virus . RCNMV MP is recruited to the VRC through the interaction with a host protein contained in the VRC  of PVX genomic RNA. The green fluorescent protein gene containing the 107 nt terminal sequence of the PVX 5′-UTR was transported effectively to neighbor cells by cobombarded PVX -coated beads, and release of bound proteins by nuclease digestion has successfully identified a number of RBPs that bind specifically to cellular mRNAs RNA viruses, translation, replication, and intra- and intercellular movements are often closely linked . To invear mRNAs . ApplicaThe 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.


Chronic hepatitis B virus (HBV) infection causes approximately 325,000 deaths from cirrhosis and liver cancer each year in the Western Pacific Region of the World Health Organization (WHO) . With anDespite having only 28% of the world’s population, approximately half of all HBV-attributable deaths globally occur in the WHO Western Pacific Region . To contAdministration of the hepatitis B vaccine birth dose followed by timely completion of the hepatitis B vaccine series is 70%–95% effective in preventing mother-to-child HBV transmission ,7. Durin*This assessment was conducted in five of 24 provinces selected on the basis of accessibility and larger population size. In each of the five provinces, the central or provincial hospitals were selected, along with a sample of two district hospitals and four health centers to ensure representation of facilities with both high and low rates of HepB-BD coverage. One district health office that offered vaccination services also was included.At each of the 37 health facilities, a standardized questionnaire was administered by in-person interview with one to three staff members, including administrators, staff members in charge of the delivery ward, and vaccination department personnel. Information collected by interview concerned staffing; job descriptions and training; policies and practices with regard to administering HepB-BD; administration of HepB-BD as part of outreach service delivery; and availability, supply, and storage of hepatitis B vaccine in the facility and for outreach delivery. Birth and vaccination registries were reviewed to obtain total birth and vaccination data for all births recorded by these facilities occurring during the 3-month period December 2011–February 2012. Data were extracted for newborns delivered in the 37 facilities and those receiving HepB-BD; among newborns receiving HepB-BD, information regarding birth setting  was obtained when available.HepB-BD vaccination was defined as administration of the monovalent hepatitis B vaccine within the first 7 days of life, per the Laos vaccination schedule. For health-care facilities providing onsite delivery services, HepB-BD coverage was calculated by dividing the number of newborns who received HepB-BD in the facility by the total number of births in that facility. The median and interquartile ranges of facility-specific coverage rates also were calculated. A total of 37 facilities known to have reported HepB-BD vaccinations in 2011 were selected to visit during the study period . These fThirty-one (84%) of the 37 facilities reported providing birthing services onsite . These 3Vaccination practices varied . Of the Of the 37 facilities visited, 17 (46%) reported assisting with home births, and 23 (62%) reported conducting postnatal home visits . Seven  focusing initial efforts on increasing coverage among newborns in health-care facilities, who are easier to access than those born at home; 2) ensuring vaccine stock availability so that HepB-BD prevention opportunities are not missed; and 3) integrating HepB-BD vaccination into ongoing home birthing and postnatal home visit services.Vaccination coverage in Laos might be improved by designating a staff member responsible for implementing HepB-BD vaccination at each facility and ensuring that birthing professionals are provided with training. Such training could include emphasizing the importance of early vaccination in preventing mother-to-child HBV infection during birth . TraininDespite the small proportion of deliveries that occur in health facilities in Laos, implementing HepB-BD vaccination in health facilities is an efficient and practical approach to preventing perinatal HBV transmission . Making Opportunities for increasing coverage to newborns born at home include integrating HepB-BD into existing maternal and newborn outreach activities. Policies supporting this activity are in place in Laos, including inclusion of HepB-BD vaccination in the recommended neonatal care package. However, outreach services remain limited because they are costly, underfunded, and highly dependent on external funding. As demonstrated in this assessment by the minimal number of home visits, outreach by a skilled provider is currently available to only a small proportion of new mothers. However, integrating HepB-BD vaccination with existing outreach activities is feasible by raising awareness, training staff members, ensuring the supply of vaccine, and taking advantage of the proven heat stability of hepatitis B vaccines, which allows for their use where refrigeration is not available .What is already known on this topic?Hepatitis B virus (HBV) infection is highly endemic in Laos, which has adopted vaccination strategies to reduce infection rates in children. A hepatitis B vaccine birth dose (HepB-BD), as part of a 3-dose schedule, is effective in preventing perinatal hepatitis B transmission. However, vaccination coverage with HepB-BD in Laos was estimated at only 34% in 2011.What is added by this report?In a survey of 37 health facilities conducted during December 2011–February 2012 in five of the 24 provinces in Laos, the median HepB-BD vaccination coverage among infants born in health-care facilities was 74%. Hepatitis B vaccine was out of stock at 49% of facilities at the time they were visited. Missed opportunities to vaccinate because of misunderstanding of vaccine contraindications and low rates of medical attendance at home births also were observed.What are the implications for public health practice?To reduce the prevalence of chronic hepatitis B infection in Laos, efforts could be directed at preventing missed opportunities for administration of HepB-BD by eliminating stock outages and dispelling misunderstandings about the vaccine among health facility staff members who attend to births. In addition, significantly improving overall birth dose coverage in the country will require ensuring inclusion of HepB-BD in home birthing and postnatal services, an increase in the proportion of home births that are medically attended, and an increase in postnatal home visits by medical staff members.The findings in this report are subject to at least three limitations. First, the assessment covered only 37 health facilities in five of the 24 provinces in Laos and cannot represent the country as a whole. Second, facility-based birth dose coverage likely is overestimated because newborns born outside of the facilities who later received HepB-BD at the facilities could not be distinguished in most instances from those born in the facility who received HepB-BD. Finally, responses on the questionnaire could not be independently verified.Laos has shown a strong commitment toward the Western Pacific Region goal of reducing chronic HBV infection prevalence in children aged ≥5 years to <1%. Such a reduction in HBV prevalence will require prevention of both perinatal and early childhood infections. Activities identified in this assessment can provide direction to help further strengthen efforts to prevent HBV infection in Laos.


Large-scale climate patterns control variability in the global carbon sink. In Europe, the North-Atlantic Oscillation (NAO) influences vegetation activity, however the East-Atlantic (EA) pattern is known to modulate NAO strength and location. Using observation-driven and modelled data sets, we show that multi-annual variability patterns of European Net Biome Productivity (NBP) are linked to anomalies in heat and water transport controlled by the NAO–EA interplay. Enhanced NBP occurs when NAO and EA are both in negative phase, associated with cool summers with wet soils which enhance photosynthesis. During anti-phase periods, NBP is reduced through distinct impacts of climate anomalies in photosynthesis and respiration. The predominance of anti-phase years in the early 2000s may explain the European-wide reduction of carbon uptake during this period, reported in previous studies. Results show that improving the capability of simulating atmospheric circulation patterns may better constrain regional carbon sink variability in coupled carbon-climate models. The relationship between terrestrial carbon sinks and atmospheric modes of variability remains uncertain. Here, the authors show that the coupling of the North Atlantic Oscillation and East-Atlantic patterns explains variations in the European CO2 sink from 1982 to 2012. Our results show that the combined NAO–EA variability explains variations in the European CO2 sink from 1982 to 2012, through their control on heat and water-vapour transport towards the continent and consequent climate anomalies. We find that the European sink is enhanced only when the two modes are in negative phase due to enhanced photosynthesis, while for both anti-phase combinations NBP is kept below average through different responses of photosynthesis and respiration.Here we analyse the combined impact of NAO and EA on NBP over the European area extending to the Ural Mountains , using t2 sink between 1982 and 2012  data, a proxy for Gross Primary Productivity (GPP)European NBP from inversions confirms that European ecosystems were on average a net COand 2012 , in lined 2012 19. The latDespite the lack of long-term NBP trend at continental scale, + years differ in NAO strength and therefore in its impacts), as well as from carryover effects from previous year's climate conditions. For example, warm and moist conditions in 1 year promote organic matter decomposition, thereby increasing nutrient availability with impact on plant growth during subsequent years+EA+, NAO−EA−, NAO+EA− and NAO−EA+), rather than individual years . In fact, both sets of NBP estimates identify NAO−EA− as the single combination with consistent estimates of NBP enhancement (except two DGVMs), even for an extended record from 1950 to 2012 (+EA− and NAO−EA+), which are significantly lower than for NAO−EA−. For NAO+EA+, MACCII inversions disagree with DGVMs and Jena inversion on the sign of the anomalies. However, the limited number of years (only four) in this composite highlights the need for caution in the interpretation of NBP anomalies for this combination. It is worth pointing that inversions present different sensitivities to NAO–EA variability: while the sign of NBP anomaly from the two MACCII versions tends to depend on the phase/anti-phase dichotomy, NBP anomalies from the Jena inversion appear to respond more strongly to EA phases.Our analysis reveals that the sole use of NAO does not suffice to explain NBP variability, because its impacts are strongly modulated by the sign of the EA. Inversions and DGVMs agree on significant differences in NBP composite anomalies during NAO− years , dependi to 2012 . During 2 uptake variability, we now propose the main components of the physical mechanisms associated, that is, linking variations in atmospheric circulation and climate variables to the anomalies observed in NBP for each NAO–EA phase combination.Having shown the need to consider both NAO and EA when assessing European CO+ (NAO−) imposing negative (positive) anomalies in the North and positive (negative) in the south, the EA modulates their strength and configuration, consistent with ; ith ref. . During + (NAO−). In contrast, during anti-phase periods, the weaker pressure gradients at the surface and in altitude leads to a northward displacement and attenuation of eastward heat flux, and water-vapour transport is close to average values in most of the continental Europe. The impact of these fluxes on cloud cover was evaluated as a means to estimate the combined variability of precipitation and sunlight, both of which are relevant (with opposing effects) for vegetation growth during these monthsBy influencing the strength and shape of the North-Atlantic jet, the interplay between NAO and EA controls the atmospheric transport of heat and humidity over the region ; colours−EA−, the inhibition of heat and water-vapour transport in winter establishes very cold conditions over most of Europe, which lead to more precipitation falling as snow rather than rain, explaining the negative soil moisture anomalies and the generally higher-than-average snow depth registered in most areas. In the Mediterranean region, soil moisture is enhanced during December–April due to the stronger advection of water vapour and prevalence of cloudiness  due to the memory effects of snow coveroudiness . In contl Europe . This sh+EA− (NAO−EA+). The patterns of soil moisture for NAO+EA− (NAO−EA+) reflect the northward displacement and attenuation of water-vapour transport anomalies: most regions register close to average winter soil moisture, with drier (wetter) winter conditions being registered only in western Russia and Iberian Peninsula. Most of central Europe registers lower (higher) levels of snow cover, followed by dry (wet) soil conditions in summer. As for NAO−EA−, the snow-depth winter patterns generally match the ones observed in summer soil moisture and temperature anomalies, although the moisture–temperature coupling is most evident for NAO−EA+. The Iberian peninsula is also affected by very low winter cloud cover during NAO+EA−, which promotes higher evaporation rates, explaining the strong water deficits in winter.The meandering SLP pattern and the corresponding energy transport during anti-phase periods impose a north-east/south-west gradient (rather than north/south as during in-phase combinations) in temperature anomalies with warmer-than-average (colder) winter/spring conditions over Scandinavia and western Russia and cooler (warmer) temperatures in the western-Mediterranean sector during NAOThe anomalies in NBP are related to two largely offsetting fluxes, photosynthesis and respiration, which respond differently to climate anomalies−EA− years in all regions, except in the Iberian Peninsula, where inversions and DGVMs diverge. The clear positive NBP anomaly during NAO−EA− cannot be explained solely by positive NDVI anomalies, since large areas in Europe present NDVI anomalies of small magnitude, and in many regions even slight browning. On the seasonal scale, the cold and dry winter and spring conditions affecting most of Europe impose negative NDVI anomalies and lower-than-average GPP  present very weak control of water availability on GPP. The enhanced CO2 uptake during the NAO−EA− phase combination is thus mostly due to enhanced GPP during the growing season, not to reduced respiration.Inversions and most DGVMs consistently estimate enhanced NBP during NAO+EA+ in most of Europe (except Iberia). However, the higher annual NDVI does not coincide with increased GPP, mainly because positive NDVI anomalies occur when radiation is sub-optimal  and hence photosynthesis is weakly stimulated. DVGMs report higher GPP during spring, which offsets the increase in respiration during these warmer conditions. In summer, NDVI is about average, however, DGVMs estimate strong negative GPP and respiration anomalies, the former being stronger than the latter, and leading the negative NBP response  report a sink reduction in all regions except western Russia, which matches the marked vegetation ‘browning' and decrease in GPP during the year  and their control on heat and water-vapour transport across Europe.−EA− conditions suggests that the highest NBP (largest uptake) should occur during periods with predominantly negative phases of both teleconnections. In fact, the largest NBP peak observed in 1996–1997 in the inversions corresponds to two consecutive years of NAO−EA−. The strong increase in NBP during 1985 also occurred during this combination of NAO and EA. In contrast, the years with lowest NBP are associated with years of opposing phases of NAO and EA: the first 3 years of the period (1982–1984) and 1998–1999, when a strong decrease in NBP is observed. Anti-phase combinations dominated the late 1990s and the 2000s, with some exceptions, with a period of 6 consecutive years of anti-phase lasting from 1998 to 2003, coinciding with a weaker sink over the European continentThe NBP enhancement during NAOst century under similar scenariosWhen computing future trends in NBP, the presence of multi-annual variability and trends in the most prominent modes of atmospheric circulation should be taken into account. In this context, anthropogenic forcing has been shown to lead to more frequent positive phases of NAOThis study shows that the non-stationary relationship between teleconnections may strongly affect the relationships found between climate and ecosystem activity. This highlights the importance of correctly reproducing the mid-latitude atmospheric circulation patterns in Earth System Models to account for inter-annual and decadal variability in the land carbon sink. More long-term high-quality observation-driven data sets are needed to further improve our understanding of the multi-year variability of the land sink and its relation to climate variability.2 inversions estimate spatial distribution of surface CO2 fluxes using a top-down approach, in which surface fluxes are estimated from atmospheric CO2 concentration measurements across the globe. An atmospheric transport model that represents the global atmospheric circulation is required, as well as prior-information about the fluxes. Flux estimates over land include the carbon exchange in ecosystems, LUC related fluxes and forest fire CO2 emissions as well as fossil-fuel emissions. To estimate NBP, the latter are subtracted from the flux estimates.Atmospheric CO2 fluxes of two versions of the atmospheric inversion of the MACCII from the LSCE292 in the planetary boundary layer, where the assimilated measurements are located.We use monthly COSince MACC inversions are based on a large but variable number of stations during the study period , with fewer observations in the beginning of the time series, part of the variability observed on the estimated fluxes may be affected by the changes in the number of sitesTo subtract fossil fuel emissions, MACCII inversions use the EDGAR3.2 FastTrack 2000 emission databaseUncertainties of annual NBP at the continental scale are given, for each model, in 2 is sampled at the same locations and times as the real data for three different station sets: as in Jena s81 (14 sites) and s90 (25 sites) as well as mostly the same sites used for MACCII inversion (2 concentrations sampled in this way were inverted back using the Jena scheme (setting ocean and fossil fuel priors to zero). As the same transport model is used for synthetic data and inversion, results are not contaminated by model errors. By comparing with the original fluxes  by transporting net ecosystem exchange (NEE) fields as simulated by the Biome-BGC DGVM in the atmosphere using the same transport model as in the Jena inversion (v3.7), though on coarser 10 × 8 spatial resolution. To create pseudo data, modelled atmospheric COnversion . The CO2l fluxes , the senDGVMs simulate the main processes of vegetation dynamics and decomposition associated with energy, water and carbon balances at the ecosystem scale and provide a bottom-up approach to evaluate NBP variability, as well as the corresponding GPP and respiration components.2 concentrations from ice core data and observations, historical climate observations from the CRU-NCEP v4 data set and LUC from the History Database of the Global Environment (HYDE) database of human-induced land-cover changesThe TRENDY projectBiweekly NDVI fields over 1982–2012 from the GIMMS NDVI data set, provided at 8 km spatial resolution1), the corresponding empirical orthogonal function (EOF1) and explained variance were selected for each inversion and model. EOF1 presented in all cases dipolar pattern. The consistency of these patterns was compared by locating the corresponding centres of action for each data set  on a pixel basis to compute monthly anomaly and integrated over the year for annual NBP anomaly fields, for the European continent and for the regional boxes . A positdata set .−1 s−1) and heat , fraction of cloud cover (%), SLP (in mb), 500-mb geopotential height , average air temperature at 2 m , volumetric soil-water content  in the top layerAll climate variables used for this analysis were extracted from ERA-Interim reanalysisAs complementary information about soil-water conditions, monthly values of the self-calibrated Palmer Drought Severity Index (PDSI)We focus our analysis on the impacts of winter state Dec-Feb (DJF) of two teleconnections, using the monthly indices from NOAA's Climate Prediction Centre, which are calculated by rotated PCA of monthly means of 500-mb geopotential height anomalies from NCEP/DOE II ref.  as descr2+ for a positive phase of winter NAO). To evaluate the combined impacts of both teleconnections, we identify four subsets of years corresponding to the four possible NAO–EA phase combinations, together with a neutral composite (The phases of an index are defined as those exceeding the lower (negative) or upper (positive) terciles, and identified in superscript  the ability of the inversion to reproduce fluxes in large regions over Europe; (ii) the influence of the observational network. Despite the very coarse resolution, results  indicateAll data fields were selected for the study region . ContineFor each composite, a one-sided analysis of variance (ANOVA) was performed to test the significance of the average anomaly value on the continental and regional scales , separatSince the synthetic runs of Jena inversion present some dependence of absolute fluxes in the observational network, it is worth assessing how the number of sites may influence the results of NBP anomalies in response to NAO–EA variations. As the observational network has increased with time, Jena inversion is provided for different periods using an increasing number  of sites. Here we compare the other two inversions from Jena v3.6 that still encompass at least 20 years and use the same model but more sites: s85 , s90 . We also performed a run with the newer version of Jena inversion (v3.7) using the MACCII sites, at the same resolution as Jena s81 v3.6. The corresponding anomalies for each NAO–EA combination are presented in How to cite this article: Bastos, A. et al. European land CO2 sink influenced by NAO and East-Atlantic Pattern coupling. Nat. Commun. 7:10315 doi: 10.1038/ncomms10315 (2016).Supplementary Figures 1-11, Supplementary Tables 1-7 and Supplementary References


The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO’s single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys319, which forms MPO’s unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO.Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single  MPO is stored in an inactive state in the azurophil granules of neutrophils, a modified type of lysosome, and becomes catalytically productive only when a neutrophil is stimulated and turns on the enzyme nicotinamide adenine dinucleotide phosphate-oxidase , 43interde bonds . We mutainter-molecular disulfide bond. At the level of immunoblot the R569W mutant resembles the C319S and double cysteine mutants in its lack of processing.Cell extracts from wildtype (wt) and mutant MPO cell lines were analyzed by immunoblot both in the absence and presence of reducing agent to assess MPO processing. The C316A mutant undergoes normal processing events indicated by levels of heterotetramer (-DTT) and cleaved heavy chain (+DTT) that is identical to those produced in wt T47D-MPO cells . Both thELISA measurements of cellular versus secreted MPO from wt and mutant cell lines show that the C316A mutant again behaved similarly to wt MPO. Both the C319S and double cysteine mutants demonstrated enhanced secretion from cells while secretion of the R569W mutant was severely decreased . The oppGlycosylation characteristics including modification with M6P were examined for the cysteine mutants. All such modifications of the C316A mutant were similar to what was observed for wt MPO in T47D cells . For theImmunolocalization of cysteine mutants in the T47D cell lines showed that the C316A mutant was localized most prominently in lysosomes and was indistinguishable from the staining pattern observed for wt MPO . The stainter-molecular disulfide bond formation is necessary for the proteolytic processing and lysosomal transport of MPO. Indeed, due to their high level of sequence identity one might have expected the C319S MPO mutant to resemble the processed hemi-dimer of eosinophil peroxidase (EPO) with no functional consequence other than the lack of the inter-molecular disulfide bond. However, increased complex oligosaccharide content, impaired trafficking, and low specific activity of the C319S mutant coupled to the absolute family conservation at C319, suggest that this position is structurally or otherwise mechanistically important for events that occur prior to diversion of MPO from the Golgi towards the lysosome. The crystal structure of the mature MPO heterotetramer clearly shows the C319 side chain is outward facing enabling it to participate in the inter-molecular disulfide bond [319 is part of an interface that interacts with the pro-domain and stabilizes the proMPO structure for events such as glycosylation that occur early in processing. Indeed, the crystal structure of (LPO) shows that the corresponding cysteine of LPO is involved in an intra-molecular disulfide bond with a cysteine that is conserved within the pro-domains of MPO, EPO, LPO and thyroid peroxidase  and just N-terminal to the homologous cysteine for LPO, means that inclusion of this additional intra-molecular disulfide bond is incompatible with removal of the MPO pro-domain at its known cleavage site. Therefore, the additional C158-C319intra-molecular disulfide bond either never forms in MPO as it does in LPO or it must undergo rearrangement in a post-ER compartment. Precedent exists for such disulfide rearrangements. Post-ER disulfide rearrangement from intra-molecular to inter-molecular has been demonstrated for Von Willebrand factor oligomerization. An intra-molecular disulfide bond formed between the Von Willebrand factor pro-domain and a domain of the mature protein is formed in the ER, but, after exiting the ER, the chaperone activity of the pro-domain is required for rearrangement of this bond prior to or concomitant with its removal by furin protease cleavage [intra to inter-molecular disulfide bond exchange of MPO is diagramed in Because of the surprising severity and complexity of the C319S mutation we are not able to definitively say whether ide bond , 6. If troxidase  which sun proMPO . In factcleavage , 47. A pMany lysosomal proteins are modified with mannose-6-phosphate (M6P), which allows them to dock with M6P-receptors (MPRs) in the trans-Golgi network and traffic to the lysosome . MPRs al4Cl that alter the pH of endosomes disrupt trafficking to the lysosome by antagonizing the binding characteristics of the cation-independent MPR (ciMPR) leading to increased extracellular secretion of cargo proteins through the constitutive secretory pathway [4Cl demonstrated that its trafficking and processing was not significantly affected by this agent, leading to the conclusion that MPO does not use MPRs to traffic to the lysosome [4Cl did not disrupt MPO trafficking to the lysosome and in fact leads to a slight decrease in the amount of secreted MPO with a concomitant increase in cellular MPO (4Cl [4Cl treatment to disrupt ciMPR function. Immunoblot comparison of cdMPR protein levels from two distinct shRNA knockdown cell lines shows similar levels of knockdown (60–75%) when compared to a negative control shRNA cell line or the parent T47D-MPO cell line  for MPO are not MPRs or a VPS10p family member. That none of these commonly used sorting receptors would be responsible for MPO trafficking is not unprecedented and non-canonical receptors have been identified for select protein cargo. The tetraspanin CD63 serves as the trafficking receptor for neutrophil elastase to azurophil granules though it has been ruled out as a candidate for MPO [To fully understand the molecular details of MPO trafficking and processing, the receptor(s) that chaperone it from the Golgi to the lysosome must be identified. Other than MPRs, members of the VPS10p family that in humans includes sortilin, SorLA, Sorcs 1, Sorcs 2 and Sorcs 3 have been shown to participate in the trafficking of lysosomal proteins and/or endocytic-dependent processing events –55. Therol cells  panel i.ol cells  panel ii for MPO . Carboxy for MPO , 57. We We describe here the in depth characterization of T47D breast cancer cells as a superior recombinant expression system for the study of the biosynthetic processing, endocytic trafficking and lysosomal storage of myeloperoxidase. Out of ten cell lines screened , only T47D and MCF7 cells processed significant quantities of proMPO into the mature MPO heterotetramer and stored it in lysosomes in a manner similar to that of MPO expressed endogenously HL60 cells. The relatedness between T47D and MCF7 cells is noteworthy. Both cell lines are derived from estrogen receptor positive breast cancers and in mRNA expression profiles of 52 breast cancer cell lines are more closely related to each other than to any other breast cancer cell line . Therefointer-molecular disulfide bond nor do these inhibitors significantly impact transport of the resulting disulfide-linked MPO dimer to the lysosome. This suggested that either the inter-molecular disulfide bond forms before separation of the heavy and light chains or that these events are mechanistically uncoupled. To better differentiate these possibilities we wanted to know whether or not blocking formation of the inter-molecular disulfide bond by mutagenesis of Cys319 would, in turn, have any effect on the proteolytic separation of the heavy and light chain. The answer we obtained from our cysteine mutagenesis study did not, however, further clarify the relationship between these events. In fact, out results led to additional questions regarding the importance of Cys319 to steps that precede its participation in the inter-molecular disulfide bond and suggested that Cys319 is important for locking proMPO into a conformation that allows both proper glycosylation and the protein-protein interactions necessary for entering the endocytic pathway that promotes these later processing events. Our data, in context with crystal structure data of LPO and MPO, led us to formulate a hypothesis that the pro-domain of MPO folds over to interact with the exposed surface of MPO containing Cys319, forming an C158-C319intra-molecular disulfide bond. Only later, after MPO has exited the ER and during pro-domain removal is the C158-C319intra-molecular disulfide bond isomerized to the inter-molecular C319-C319 bond that links together the two heavy chains in the heterotetramer  Fluorescent images of the T47D-MPO stable cell line labeled with antibodies against MPO (green) and the ER marker KDEL (red). Colocalization is indicated by the arrowhead. (B) Fluorescent images of the MCF7-MPO cell line labeled with antibodies against MPO and Lamp1 (upper panel), the early endosome marker EEA1 (middle panel) and ER marker KDEL (bottom panel) with colors as indicated in each panel. (C) Fluorescent images of the MDA468-MPO cell line labeled with antibodies against MPO (red) and Lamp1 (green). (D) Neuro2A-MPO cell line labeled with antibodies against MPO (red) and Lamp1 (green). MPO staining concentrated in the termini of cell processes is indicated by arrowheads. The termini of Neuro2A cells contain regulated secretory granules that are distinct in origin from lysosome granules. Blue color represents DAPI staining of nuclei in all color images.(TIFF)Click here for additional data file.S2 Fig(A) Cells grown on coverslips were double-labeled with the indicated antibodies and imaged with a 63x oil objective on a Zeiss LSM 710 confocal microscope. (A) Fluorescent images of the T47D C316A-C319S double mutant cell line labeled with goat polyclonal antibodies against MPO (red) and a rabbit antibody against the early endosome marker EEA1 (green) (upper panel) or Mab-16E3 against MPO (red) and rabbit antibody against the trans-Golgi marker RCAS1 (green) (lower panel). (B) Cell extracts derived from T47D stable cell lines expressing wt or mutant MPO were incubated on duplicate ELISA plates coated with multi-epitope rabbit polyclonal anti-MPO antibody. Bound MPO was detected either with HRP-conjugated Mab-16E3 or with an HRP-conjugated multi-epitope goat polyclonal antibody. Both Mab-16E3 and the goat polyclonal detection antibodies yield identical measurements of MPO concentration for wt MPO and the cysteine mutants, whereas binding of Mab-16E3 to the R569W mutant is significantly impaired relative to the goat polyclonal. Assay points were in triplicate and plotted as the mean ± SE. Results are representative of two independent experiments. Data for each cell line was normalized to the highest value before plotting to compensate for different expression levels between cell lines.(TIFF)Click here for additional data file.


The aim of this study is to identify independent pre-transplant cancer risk factors after kidney transplantation and to assess the utility of G-chart analysis for clinical process control. This may contribute to the improvement of cancer surveillance processes in individual transplant centers.1655 patients after kidney transplantation at our institution with a total of 9,425 person-years of follow-up were compared retrospectively to the general German population using site-specific standardized-incidence-ratios (SIRs) of observed malignancies. Risk-adjusted multivariable Cox regression was used to identify independent pre-transplant cancer risk factors. G-chart analysis was applied to determine relevant differences in the frequency of cancer occurrences.2 , ADPKD  and diabetic nephropathy . G-chart analysis identified relevant changes in the detection rates of cancer during aftercare with no significant relation to identified risk factors for cancer-free survival (p<0.05).Cancer incidence rates were almost three times higher as compared to the matched general population . Significantly increased SIRs were observed for renal cell carcinoma (SIR = 22.46), post-transplant lymphoproliferative disorder (SIR = 8.36), prostate cancer (SIR = 2.22), bladder cancer (SIR = 3.24), thyroid cancer (SIR = 10.13) and melanoma (SIR = 3.08). Independent pre-transplant risk factors for cancer-free survival were age <52.3 years : 0.82), age >62.6 years , polycystic kidney disease other than autosomal dominant polycystic kidney disease (ADPKD) , high body mass index in kg/mRisk-adapted cancer surveillance combined with prospective G-chart analysis likely improves cancer surveillance schemes by adapting processes to identified risk factors and by using G-chart alarm signals to trigger Kaizen events and audits for root-cause analysis of relevant detection rate changes. Further, comparative G-chart analysis would enable benchmarking of cancer surveillance processes between centers. Kidney transplantation has become the preferred treatment option for patients with renal failure since the first successful clinical transplantation in 1954 . The sucLong-term cancer risk is known to vary significantly depending on ethnicity, demographic variations and geographical epidemiological differences in the prevalence of viral diseases with associated increased cancer risks . The detG-charts are based on the geometric distribution and were designed to monitor rare events. In health care they have initially been developed to monitor and illustrate cardiac bypass infections, catheter-associated infections, surgical site infections, contaminated needle sticks, osteomyelitis treatment failures and medication errors –35. WhilThe current study determines site-specific cancer incidence rates in patients after kidney transplantation compared to an age- and sex-matched general reference population in Germany and characterizes independent pre-transplant risk factors for cancer-free survival with possible implications for individualized long-term cancer surveillance. Findings are used to explore the principal suitability of G-chart analysis to identify relevant changes in cancer detection rates with the goal to develop a strategy for improvement of aftercare.All consecutive kidney transplants from the 01.01.2000 until the 31.12.2012 with follow-up in our adult outpatient clinic were analyzed. We did not exclude 40 pediatric cases who have reached age 18 years prior to their first follow-up visit after transplantation in our adult transplant outpatient clinic because we feel that they were managed exactly the same way as all other adult patients. Combined transplants, re-transplants, and cases with 30-day post-transplant mortality were excluded. Non-melanoma skin cancers were not analyzed since they are not registered in cancer registries.This is a retrospective single-center analysis with 9,425 person-years of follow-up until the 18.02.2014  with observational endpoints being either first diagnosis of malignancy, death, graft failure or the date patients were last seen alive. All patients were followed in our outpatient transplant clinic as described before .According to the Professional Code of the German Medical Association (article B.III. § 15.1) neither informed consent nor approval of an ethics committee was needed. The Ethics Committee of Hannover Medical School approved of this retrospective study . Patients provided informed consent that their data may be used for scientific purposes at the time of hospital admission which is the general policy of our institution. Patient records and patient data were anonymized and de-identified prior to analysis. None of the transplant donors were from a vulnerable population and all donors or next of kin provided written informed consent that was freely given.1655 patients  and 672 females (40.6%)) with a mean age of 50.0 years at transplantation  met the inclusion and exclusion criteria. 40 patients (2.4%) were younger than 17 years at transplantation and classified as pediatric patients due to the application of different donor organ allocation rules until the age of 17. Indications for renal transplantation are summarized in Missing data was below 5% for all examined variables. Cases with missing data for variables examined in univariable and multivariable Cox regression were excluded.Site-specific cancer incidence rates in the cohort were compared to the general German population using SIRs (SIR = observed incidence/expected incidence) with 95%-confidence-intervals (95%-CI) as described before . PubliclUnivariable and risk-adjusted multivariable Cox regression analyses were used to determine independent pre-transplant risk factors for cancer-free survival. Cancer-free survival was defined as survival until death or the first diagnosis of de novo malignancy and measured until whichever incident was noted earlier.After exclusion of multicollinearity in principal component analysis, variables with p-values ≤0.200 in univariable Cox regression analysis were included into an initial multivariable Cox regression model deploying purposeful variable selection. After the exclusion of significant two-way variable interactions, only those co-variables in the most parsimonious model were considered for inclusion into the preliminary Cox regression model, using a stepwise backward elimination method .Assessment of geometrical distribution of the probability of malignancy diagnosis dates was carried out under the assumption that the number of opportunities for cancer detection was fairly stable using the probability density function (p = 0.028). This assumption appeared justified due to the relatively stable amount of annually performed transplants .G-chart analysis was used to illustrate the time at risk in days between consecutive dates of actual observations of post-transplant malignancy. The center line (CL), the upper control limit (UCL) and the lower control limit (LCL) were calculated and defined to demonstrate the central tendency and the range of natural variation of the plotted values , 34, 36.The application of probability-based control limits reduced the false alarm rate and improved the capability to detect process changes , 34, 36.The Benneyan test was deployed to identify a statistically relevant number of values below or at the LCL set to zero. Test 1 was deployed to detect relevant decreases in diagnostic event rates when times between events exceed three standard deviations with a probability of being above the UCL < = 0.0013499 , 34, 36.2, Mann-Whitney-U tests and logistic regression were used where appropriate. For all statistical tests a p-value <0.05 was defined as significant. The statistical software packages Minitab 17  and JMP version 11.0  were used.Two-sided Chi152 de novo malignancies were observed in 144 patients  . The cumulative incidences of malignancies 5 and 10 years after transplantation were 5.2% and 8.2%, respectively. The SIR for all de novo malignancies was significantly increased (SIR = 2.75) . S2 Tabl2) and were on average 10 years younger without reaching statistical significance .The risk for renal cell carcinoma (RCC) was significantly increased after transplantation as compared to the general population . 37 pati20 patients experienced prostate cancer. The risk for prostate cancer after transplantation was significantly increased as compared to the general population . The meaFive patients experienced bladder cancer. The risk for bladder cancer was significantly increased as compared to the general population . PatientThe risk for PTLD/NHL was significantly increased as compared to the risk for NHL in the general population  with a tThe risk for thyroid cancer after transplantation was significantly increased as compared to the general population . All sevThe risk for malignant melanoma was significantly increased . The meaAutosomal dominant polycystic kidney disease (ADPKD), polycystic kidney disease other than ADPKD (PKD), and diabetic nephropathy were significantly associated with cancer-free survival after kidney transplantation in univariable Cox regression analysis . Transpl141 out of 1555 patients undergoing dialysis prior to transplantation developed post-transplant de novo malignancies . The duration of pre-transplant dialysis did not significantly influence the risk of cancer development during follow-up . Preempt17 out of 411 patients transplanted under the age of 39.9 years (1st quartile) presented with post-transplant de novo cancer while the incidence of malignancy was higher in the age group older than 62.6 years at transplantation (4th quartile). Age groups in quartiles demonstrated a differentiated influence on cancer free-survival .17 out of 115 patients with a history of cancer prior to transplantation developed de novo malignancies . There w2). Furthermore, univariable Cox regression analysis revealed a significant influence of body mass index and insulin-dependent diabetes on cancer-free survival  indicating an unusually low rate of detected de novo malignancies. 22 cases failed the Benneyan test indicating relevant increases in de novo malignancy detection rates . G-chartThis study is unique as it provides the distribution of tumor stages of de novo malignancies in comparison to cancers in the general population and identifies underlying causes of end-stage renal failure as independent risk factors for cancer-free survival.This is the first study that reports SIRs in kidney transplant recipients from Germany with additional analysis of independent risk factors for cancer-free survival. These incidence rates were almost three times as high as compared to the age- and sex-matched general population (SIR 2.75). Higher incidence rates have been reported from Australia and New Zealand (SIR 3.27)  indicatiAs in previous studies with non-melanoma skin cancer excluded, RCC was the most commonly observed cancer entity. The SIR of RCC was higher in this study  as compaIn contrast to previous studies , 40–41, Surprisingly and in clear contrast to our previously published observations concerning liver transplantation , vulva cPatients with de novo malignancies were approximately 10 years younger in this study than patients with comparable malignancies in the general population . FrequenBody mass index was identified here as an independent risk factor in multivariable analysis. Obesity has been reported to promote tumor growth in non-transplanted patients . SeveralFurthermore, age was identified as independent risk factor for cancer development after transplantation. Various factors such as the degeneration of DNA, mutations, aberrant replication and apoptosis leading to an increased tumor growth have been identified in previous studies, independently of kidney transplantation , 47.Interestingly, PKD was identified as an independent protective factor while ADPKD was an independent risk factor for cancer-free survival. A very recent study also described a lower cancer incidence in renal transplant patients with PKD as compared to transplanted patients without PKD . The reaDiabetic nephropathy emerged as an independent risk factor for cancer-free survival. Diabetes per se is known to promote tumor-growth . This obDue to frequent changes in dosages and types of immunosuppressive medication during follow-up it was not possible to analyze the impact of the complete immunosuppressive therapy on de novo cancer risk systematically. We believe that an analysis of the complete immunosuppressive medication over time which takes into account all changes of dosages, medication and immunosuppressants’ blood levels would be required.Pre-transplant risk factors for de novo cancer have not been well described before while post-transplant risk factors for de novo cancer such as ATG induction, mTOR inhibitors, rejection and EBV status are already known and have been described in several previous publications. The definition of post-transplant risk factors for the development of de novo cancer after kidney transplantation was not the goal of the current study. The main goal of the current study was to explore the potential of G-chart analysis for process improvement and benchmarking of post-transplant cancer surveillance during ongoing post-transplant management. The independent pre-transplant risk factors described in this study help to inform the post-transplant management process prior to and immediately after transplantation. These pre-transplant risk factors further inform the interpretation of ongoing prospective G-charts with the aim to improve post-transplant process control as early as possible. The identified pre-transplant risk factors may help to adapt clinical post-transplant management individually for patients with pre-transplant risk factors for de novo cancer, e.g. by choosing different types and intensities of immunosuppression.We believe that the results of this retrospective study show the principal value of G-chart analysis for the early identification of relevant increases and decreases in de-novo malignancy detection rates. Several advantages of G-charts in comparison to standard Shewhart-charts need to be addressed. First, G-charts are able to provide useful results even if the availability or frequency of the plotted values is low, which is the case for de-novo malignancies. Conventional charts such as n, np or c-charts will need a certain number of values to detect a significant process change. This can necessitate waiting until the end of longer time periods to calculate and interpret the results . In suchProspective deployment of G-chart analysis may identify alarm signals in case of significantly decreased or increased cancer detection rates. This allows early root cause analysis, for example by conducting a Kaizen event and additional statistical analyses including re-evaluation of independent risk factors for cancer-free survival. For a Kaizen event clinical managers and process owners as well as transplant experts are gathered to map the current process in a deployment flow chart looking for process improvements and possible root causes for unusually high or low defect rates . VaryingProspective G-charts could be used to facilitate visual management in outpatient clinics with the goal to increase the awareness and alertness in regard to cancer risks after transplantation. This may ultimately lead to improved cancer detection or even prevention in transplant patients , 34. As We accept the methodological limitations of the current study including the lack of timely root cause analysis for the G-chart findings which is owed to the retrospective nature of this study. This also implies that there is a possibility of underreporting of cancer. Further limitations of this study are caused by the small sample size of some cancer types and primary diseases and the fact that non-melanoma skin-cancer, the most common form of cancer in transplanted patients, were not analyzed. Furthermore, a likely center bias has to be taken into account. Altogether, we believe that this study clearly demonstrates a powerful methodological approach to the improvement of cancer surveillance management in transplant aftercare at the transplant center level providing a perspective for transplant center benchmarking.This study determined site-specific cancer incidence rates after kidney transplantation, showing that de-novo malignancies are a current and important issue to be addressed. Furthermore, this study showed that certain pre-transplant characteristics play an important role in terms of de-novo malignancies after transplantation. The usage of G-chart analysis during post-transplant management can improve cancer surveillance processes by triggering Kaizen events.S1 TableShown are the causes of end-stage renal failure with ≥ 10 cases leading to kidney transplantation in adult and pediatric recipients of the study-population.(DOCX)Click here for additional data file.S2 TableShown are the 5-year survival rates  for patients after diagnosis of de novo malignancy after kidney transplantation with significantly increased SIRs in the current study in comparison to 5-year survival rates of cancer patients reported for the general German population by the Robert Koch-Institute, Berlin in 2010 (n.a. = not applicable).(DOCX)Click here for additional data file.S3 TableShown is the time interval between renal transplantation (RTX) and the diagnosis of different de novo malignancies with significantly elevated SIRs in years and the mean patient ages at the time of diagnosis of cancer in the general population and after kidney transplantation. * Brackets show mean ages at diagnosis of in situ tumor stages and neoplasms with unknown or uncertain characteristics. .(DOCX)Click here for additional data file.


In ex vivo experiments, this effect could be attenuated by targeting CTLA-4 on Tregs. Therefore it is possible that the clinical efficacy of CTX may be improved by strategies to enhance the activation of the immune system or inhibit the Treg suppressive effects.HNC is immunosuppressive, with low absolute lymphocyte counts, impaired activity of effectors such as natural killer cells (NK), and poor antigen-presenting function. The anti-EGFR monoclonal antibody cetuximab (CTX) prolongs survival in HNC, but only in a subset of patients. The clinical activity of CTX has been mechanistically linked to NK-mediated antibody-dependent cellular cytotoxicity (ADCC). However, recent data has shown that CTX increases the frequency of intratumoral CTLA-4Motolimod (formerly VTX-2337) is a novel Toll-like receptor 8 (TLR8) agonist which stimulates myeloid dendritic cells (mDC), monocytes, and NK. Preclinical data have demonstrated enhanced CTX and NK-mediated lysis of HNC cells and dendritic cross-priming of EGFR-specific CD8+ T cells. CTX and motolimod in HNC patients was tolerable and active in a Phase Ib study, with enhanced mobilization and activation of NK cells. The central hypothesis in this study is that NK and monocyte/mDC activation by CTX is enhanced by concomitant administration of motolimod, thereby amplifying the innate and adaptive immune response in the circulation and in the tumor microenvironment.This is a prospective Phase Ib clinical trial (NCT02124850) of preoperative treatment with CTX and motolimod. The primary objective is to determine the extent to which the administration of neoadjuvant CTX plus motolimod modulates immune biomarkers in peripheral blood and HNC tumors. An exploratory objective of the study is to assess whether this modulation of biomarkers can predict anti-tumor response. Subjects must have HNC stage II– IVA and be surgical candidates. CTX and motolimod will be administered for a 3–4 week preoperative period. The biomarker modulation and biopsies of skin/acneiform rash will be studied in correlation with clinical response by CT or MRI scan. Tumor apoptosis/proliferation will be assessed by biopsy pre- and post-treatment with CTX/motolimod. All patients will receive definitive surgical resection. Enrollment target is fifteen complete specimens . A pending amendment will assess the impact of adding checkpoint inhibition to the CTX/motolimod combination.