The ability to recognize foreign double-stranded (ds)DNA of pathogenic origin in the intracellular environment is an essential defense mechanism of the human innate immune system. However, the molecular mechanisms underlying distinction between foreign DNA and host genomic material inside the nucleus are not understood. By combining biochemical assays and single-molecule techniques, we show that the nuclear innate immune sensor IFI16 one-dimensionally tracks long stretches of exposed foreign dsDNA to assemble into supramolecular signaling platforms. We also demonstrate that nucleosomes represent barriers that prevent IFI16 from targeting host DNA by directly interfering with these one-dimensional movements. This unique scanning-assisted assembly mechanism allows IFI16 to distinguish friend from foe and assemble into oligomers efficiently and selectively on foreign DNA.DOI:http://dx.doi.org/10.7554/eLife.11721.001 The immune system defends us from attacks by viruses, bacteria and other microbes. One way that the immune system can identify these invaders is by detecting genetic material from the microbes. Like us, many of these microbes have genetic material made of a two-stranded molecule called DNA. A protein called IFI16 is an important sensor in immune cells that can bind to foreign DNA, but not to the DNA of the host. If the ability to discriminate between host and foreign DNA is lost, then immune cells start to attack the body’s own tissues, which can lead to severe “autoimmune” diseases. However, it is not clear how IFI16 and other sensor proteins are able to tell foreign and host DNA apart.DNA in host cells is packaged in particular proteins to form structures called nucleosomes that can make it difficult for other proteins to bind to the DNA. When foreign DNA enters the cell, IFI16 promotes the formation of nucleosomes on these molecules, but the nucleosomes are further apart than those in host DNA, which leaves longer stretches of “exposed” DNA between the nucleosomes. In 2014, a group of researchers reported that multiple molecules of IFI16 associate with each other and form clusters on exposed foreign DNA. Here, Stratmann, Morrone et al. – including some of the researchers from the earlier work – investigated how IFI16 selectively binds to foreign DNA using a combination of biochemical and single-molecule techniques.The experiments show that to start with, a few molecules of IFI16 bind to stretches of exposed foreign DNA and then move along the DNA strands. As more molecules of IFI16 bind to this DNA, they bump into each other and start to form clusters that eventually become immobile. Further experiments show that the more tightly packed nucleosomes in host DNA molecules act as a barrier to IFI16 cluster formation because they interfere with the ability of the protein to move along the DNA.Stratmann, Morrone et al.’s findings show that IFI16 can only trigger immune responses when it binds to stretches of exposed DNA that are long enough to allow the assembly of IFI16 clusters. The next challenge will be to see whether other DNA sensors employ similar strategies to detect foreign DNA.DOI:http://dx.doi.org/10.7554/eLife.11721.002 The host innate immune system detects infection by directly recognizing molecular signatures associated with pathogens . Remarka5 to 106 base pairs (bps). Second and more importantly, this assembly mechanism can only take place on foreign dsDNA and must be inhibited on host dsDNA (The interferon-inducible protein 16 (IFI16) is a key innate immune sensor that detects foreign dsDNA and uses it as a scaffold to assemble supramolecular signaling platforms in both the host nucleus and cytoplasm  Figure . IFI16 pst dsDNA . Here, wTo identify the mechanisms underlying assembly of IFI16 signaling platforms on DNA, we monitored the oligomerization kinetics of FRET donor and acceptor labeled IFI16 on naked dsDNA  as a C-terminally His6-tagged protein as described in Morrone et al.  as described in Morrone et al. . The comDyLight-550, DyLight-650, or Cy5 fluorophore was incorporated to IFI16 using maleimide chemistry (purchased from Thermo Scientific and Invitrogen) and was performed as described in Morrone, et al. . The labLyophylized Xenopus laevis histones H1A, H2A, H3, and H4 were a kind gift of Dr. Cynthia Wolberger. Octamer refolding and nucleosome reconstitution was performed as described in Luger et al. , at a 2:All absorption, fluorescence anisotropy, and fluorescence excitation/emission experiments were performed in a Tecan Infinite M1000. All experiments were performed at least three times and the fits to data were generated by Kaleidagraph software (synergy).All reactions were performed in 40 mM HEPES pH 7.4, 160 mM KCl, 5% glycerol, 1 mM EDTA, 0.1% triton-X-100, 5 mM DTT (Reaction Buffer). Here, 300 nM IFI16 and 4.5nM fluorescein-labeled dsVACV72 were incubated together at room temperature for 20 min. Increasing concentrations of competing DNA were added to the reaction to a final concentration of 100 nM IFI16 and 1.5 nM dsVACV72, and the changes in fluorescence anisotropy were recorded as indicated in Morrone et al. .All reactions were performed in reaction buffer. 66 µg/ml of each dsDNA or di-nucleosomes was placed in the plate wells, and the reaction was initiated by adding an equivalent volume of IFI16-550 and IFI16-650 (1:1 molar ratio) to the indicated final concentration. The dead time between addition of IFI16 and the first measurement was 15-20 s. The final dsDNA molar-concentrations are at least sixfold higher than their determined binding constants by fluorescence anisotropy assays described in Morrone, et al. , and theMicroscope coverslips (Corning) were plasma-cleaned and activated with 100 mM KOH, silanized with 3-Aminopropyl-triethoxysilane in acetone and functionalized with PEG-NHS and biotin-PEG-NHS (Laysan-Bio) in sodium bicarbonate with a 1:3 mass ratio . StreptaSingle-molecule assays were performed in 40 mM HEPES (pH 7.4), 160 mM KCl, 1 mM EDTA, 2 mM DTT, 10% glycerol, 0.1% Triton-X100, 250 µg/ml BSA, 1 mM Trolox, 40 mM glucose, 250 nM glucose oxidase, 60 nM catalase, unless otherwise stated. All assays were performed at room temperature. We applied 100 nM YoPro-1 iodide (Life Technologies) at the end of the measurements to visualize the DNA substrates.Reactions were illuminated with a 488-nm and 641-nm laser (Coherent), and images were acquired with an EMCCD camera (Hamamatsu). We used MetaVue imaging software (Molecular Devices) for data acquisition and ImageJ and R for analysis.Lambda-DNA (New England Biolabs) was biotinylated at one or both ends by ligation of the respective complementary biotinylated oligos according to Tanner et al.   were assembled on biotin-λ-DNA and biotin-601 sequence (Epicypher) by salt-gradient dialysis  in 10 mM Tris/HCl, pH 7.4, 0.1 mM EDTA, 0.5 mM DTT. We tested nucleosome reconstitution by an EMSA assay on digested λ-DNA . For thiAntibodies against human histone H4 and H2B were chosen to target the N- terminal histone tails , and labeled with Atto488-NHS (Life technologies) in PBS at pH 7.0. Labeled antibodies were negatively tested against bare DNA and Ifi16 clusters in the microscopy assay, and showed high specificity for nucleosomal DNA preparations only.λ-DNA templates, loaded with nucleosomes and tagged with anti-H4, were constantly stretched in the flow channel. Ifi16 co-localized strongly with the nucleosome signal . In contFor the clustering experiments, we applied a flow of 0.02 ml/min to our flow cell design of 0.1 mm height and 1 mm width, giving a volumetric flux of 0.33 cm/s. We expect the DNA molecules to be on average 0.2 µm away from the surface , giving The Stokes drag force that acts on the DNA and bound IFI16 molecules is then described by η, radius r and distance y.with viscosity We can define this force within the diffusion coefficient D by using the drift velocity v, calculated from single-molecule trajectories :with v=∑2/s) to allocate each other on a λ-DNA molecule congested with a varying amount of other diffusing IFI16 molecules . As all particles are equal, we segmented the DNA according to the total number of molecules bound and calculated the effective distance between two particles by using the absolute distance modulo the segment length in order to take the periodic boundaries into account.In order to evaluate the effect of flow on the clustering mechanism, we implemented a 1D-random walk Monte-Carlo simulation . Here, wvdt was added  and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled "The innate immune sensor IFI16 recognizes foreign DNA in the nucleus by scanning along the duplex" for consideration by The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Summary:The manuscript reports on single molecule tracking studies of the IFI16 DNA sensor on DNA molecules without or without loaded nucleosomes. The work shows that IFI16 can diffuse along the naked DNA and form oligomers. Movement along the DNA is impeded by nucleosomes if spaced closely enough and oligomer formation is reduced. The authors conclude that IFI16 scanning is required for oligomer formation. This is a very timely and potentially important piece of work.Essential revisions:The reviewers felt there were a few areas that needed strengthening:1) The connection of scanning to IFI16 function is probably beyond the scope this manuscript, but the text needs to more clearly state that IFI16 scanning on DNA could contribute to both innate signaling and epigenetic silencing initiated by IFI16 binding to DNA.2) The reviewers all felt that the conclusion that scanning was required for oligomer formation needed further documentation or was too strong. One reviewer felt that additional biophysical studies were needed to relate scanning to oligomer formation, while the other two had questions about the specific interpretation of the results. For example, the question of how the binding stoichiometry of IFI16 relates to the two HIN DNA-binding domains was considered and what is the role of each of the HIN domains? How were the data interpreted to allow the conclusion that scanning is required for oligomer formation as opposed to a parallel process to cooperative binding? How was the lower limit of the number of IFI16 molecules per immobile cluster determined? Much of this involves revising the manuscript for a more general audience but may require some additional experiments.3) There are some minor issues on referencing as addressed below that should be addressed.There are a number of details that are embedded in the individual reviews so we are including those so the authors can understand the issues more fully. The areas that need to be addressed are summarized above.Reviewer #1: This manuscript reports on single molecule tracking studies of the IFI16 protein on dsDNA. Consistent with previous studies from the Sohn lab, IFI16 binding to DNA requires large DNA fragments involving oligomers of IFI16. To examine the mechanism of assembly of the oligomers, they tracked single molecules of IFI16 on stretched DNA. They observed that IFI16 tracks along stretches of DNA and they interpret this as required for assembly of the oligomers. Addition of nucleosomes to the DNA at sufficiently high density prevented IFI16 from moving and oligomerizing.eLife should be written for the general reader.The scanning of IFI16 along DNA to assemble oligomers to initiate signaling for either innate responses or epigenetic silencing is an important observation. The definition of a mechanism by which IFI16 distinguishes between foreign and self DNA is an important result as well, so this is potentially an important piece of work for the field. I have one reservation. It is not clear from the presentation how the results demonstrate that scanning is required for the assembly of the oligomers versus just happening in parallel to cooperative binding. The text needs to better define the reasoning and interpretation of the results to allow this conclusion. This may be in part due to a lack of background of this reviewer, but manuscripts for For example, in the fourth paragraph of the main text it is not clear how the lower limit of the number of IFI16 found in immobile clusters was determined to be 8.I have a few minor citation issues as well:1) Main text, second paragraph. Johnson et al., 2013 is really derivative of the other earlier references, which should include Orzalli et al., 2012.2) Main text, second paragraph. IFI16 is associated with autoimmunity, but it not known how it "promotes" it.3) Main text, sixth paragraph. This states that the chromatin inhibition hypothesis has not been tested directly. Orzalli et al., 2013 did compare gene expression of SV40 DNA in chromatin versus free DNA.Reviewer #2: In this manuscript, Stratmann et al. used a combination of bulk biochemistry and single molecule analysis to investigate the filament assembly process of IFI16 on dsDNA, and the mechanism by which IFI16 measures the dsDNA length as a basis for self vs. non-self-discrimination. Through FRET kinetics analysis, the authors showed that IFI16 binds to dsDNA in a length dependent manner , and drew the conclusion that 10 protomers are necessary to form the stable filament nucleus. They then used TIRF microscopy to show that IFI16 can diffuse on DNA under flow and proposed that this facilitates filament assembly and DNA length measurement. The authors also used the nucleosome coated DNA to show that IFI16 can only diffuse within the nucleosome-free region of DNA, and proposed that the nucleosome structure limits its filament assembly on self DNA.The proposed model of DNA sensing by 1D diffusion is interesting and novel. However, I have a number of concerns. In particular, the authors failed to show the importance of diffusion in filament assembly, which is the main conclusion of this manuscript.The length sensitivity observed between ~50 bp-200 bp in Another reason that makes me doubt about the importance of diffusion is the range of DNA length that IFI16 appears to sense. The authors observed the assembly rate increases with the DNA length but plateaus around ~200 bp. Since the authors clearly showed that IFI16 can diffuse far beyond 200 bp (>2-3 kb), this observation cannot explain the observed saturation at ~200 bp. On the other hand, this can be simply explained by the requirement of ~10 protomers to form a stable filament, as suggested by the authors. Note that there are proteins that form filaments in a nucleic acid length-dependent manner but without 1D diffusion. If diffusion is important for filament assembly, there should be a length dependence beyond 200 bp, especially at low protein concentration where the 3D diffusion-mediated filament assembly is significantly compromised. Again, without kinetic modeling that clearly shows the inconsistency with 3D diffusion and requirement for 1D diffusion, it is hard to arrive at the same conclusion as the authors.Another concern is that the 1D diffusion was shown only under flow. I understand that this is required to visualize diffusion (as you need to stretch the DNA), but this makes diffusion mostly uni-directional, and could likely affect the scanning processivity and the contribution of diffusion to filament assembly efficiency. More importantly, long DNA  collapses in the absence of flow, in which case 3D diffusion (or hopping), in comparison to 1D diffusion, could be more important for the filament assembly.In summary, while the authors showed 1D diffusion under flow and length sensitivity in bulk biochemistry, I don't think their data suggest that 1D diffusion is required for the length sensitivity and filament assembly.Reviewer #3: This is a timely and important study that addresses a fundamental question in immune response: How does a nuclear DNA sensor recognize pathogenic dsDNA from host dsDNA within the same subcellular compartment , a recognition critical for the initiation of immune signals? The study focuses on the nuclear DNA sensor IFI16, the interferon inducible protein 16. The authors used fluorescence assays to investigate the binding of IFI16 to foreign dsDNA, first measuring the number of IFI16 molecules necessary for assembly. Using single-molecule fluorescence imaging, the authors go on to monitor the diffusion of IFI16 along lambda-phage dsDNA. Lastly, as chromatinization was previously proposed as a critical contributor to the ability of IFI16 to distinguish between host and foreign dsDNA, the authors perform competition-binding assays and time-resolved fluorescent assays to gain further insight into the impact of chromatinization on pathogenic DNA sensing. Overall, this study elegantly incorporates fluorescence imaging assays to address current questions relevant to the initiation of intrinsic and innate immune responses. This study can have a broad impact on understanding mechanisms of host defense against nuclear-replicating DNA viruses.1) Based on the FRET results shown in 2) The authors propose that IFI16 scans along dsDNA  and 2. H3) The authors show beautifully the movement of IFI16 along the lambda-phage dsDNA and propose that this diffusion is critical for IFI16-mediated immune responses. However, it is not clear if this movement is a property specifically required for triggering immune response rather than a property involved in the transcriptional regulatory functions of IFI16. Are the immobile clusters sufficient for triggering immune response? The authors could add to their Discussion to address these possibilities. The reviewers felt there were a few areas that needed strengthening:1) The connection of scanning to IFI16 function is probably beyond the scope this manuscript, but the text needs to more clearly state that IFI16 scanning on DNA could contribute to both innate signaling and epigenetic silencing initiated by IFI16 binding to DNA.Our results indicate that one-dimensional diffusion allows IFI16 to assemble into distinct clusters specifically on naked or underchromatized DNA of several kbp length. These clusters are seen as the active “firing” form, because the oligomerized PYD domains are thought to be the platform for downstream signaling . In the revised manuscript, we expanded the Discussion to highlight this aspect.We are aware of the recent literature on epigenetic silencing that might be initiated by IFI16 as well (e.g. [3]). It is difficult, however, with our in vitroassays to derive a possible epigenetic silencing mechanism mediated by IFI16. However, we revised the discussion in our revised manuscript to raise this possibility.2) The reviewers all felt that the conclusion that scanning was required for oligomer formation needed further documentation or was too strong. One reviewer felt that additional biophysical studies were needed to relate scanning to oligomer formation, while the other two had questions about the specific interpretation of the results. For example, the question of how the binding stoichiometry of IFI16 relates to the two HIN DNA-binding domains was considered and what is the role of each of the HIN domains? How were the data interpreted to allow the conclusion that scanning is required for oligomer formation as opposed to a parallel process to cooperative binding? How was the lower limit of the number of IFI16 molecules per immobile cluster determined? Much of this involves revising the manuscript for a more general audience but may require some additional experiments.These are all important questions and we are grateful for the reviewers to have raised them. Below, we will discuss these issues one by one. In summary, we obtained assembly rate constants as a function of protein concentration to exclude a major contribution of cooperativity, and introduced new single-molecule analyses to support the same assertion. We calculated the effect of flow on the one-dimensional diffusion of individual IFI16 proteins and show it to be negligible. Finally, we inserted additional information and explanation in our manuscript to better address a number of the issues raised by the reviewers.3) There are some minor issues on referencing as addressed below that should be addressed. There are a number of details that are embedded in the individual reviews so we are including those so the authors can understand the issues more fully. The areas that need to be addressed are summarized above.Further detailed below.Reviewer #1:This manuscript reports on single molecule tracking studies of the IFI16 protein on dsDNA. Consistent with previous studies from the Sohn lab, IFI16 binding to DNA requires large DNA fragments involving oligomers of IFI16. To examine the mechanism of assembly of the oligomers, they tracked single molecules of IFI16 on stretched DNA. They observed that IFI16 tracks along stretches of DNA and they interpret this as required for assembly of the oligomers. Addition of nucleosomes to the DNA at sufficiently high density prevented IFI16 from moving and oligomerizing. The scanning of IFI16 along DNA to assemble oligomers to initiate signaling for either innate responses or epigenetic silencing is an important observation. The definition of a mechanism by which IFI16 distinguishes between foreign and self DNA is an important result as well, so this is potentially an important piece of work for the field. I have one reservation. It is not clear from the presentation how the results demonstrate that scanning is required for the assembly of the oligomers versus just happening in parallel to cooperative binding. The text needs to better define the reasoning and interpretation of the results to allow this conclusion. This may be in part due to a lack of background of this reviewer, but manuscripts for eLife should be written for the general reader.The reviewer raises a very important issue, one that was also brought up by reviewer #2. To address this concern, we performed experiments in which we use solution-based FRET to monitor the assembly rate of IFI16 aggregates on DNA for different protein concentrations.As shown in Furthermore, as shown in Author response table 1 below, the calculated Hill constants from our previous FRET assays that monitor the equilibrium assembly of IFI16 oligomers are generally below two for dsDNA sizes ranging from 60 to 2000 bps , the intensity values of individual proteins form a narrow distribution, whose mean value can be used to ‘count’ the number of proteins in a cluster. We obtained the mean intensity value for single IFI16 molecules by analyzing those that bind transiently to the lambda DNA, and calculated from this mean intensity value  the molecules in clusters that form at the critical concentration of 3 nM. We added the intensity histogram in I have a few minor citation issues as well: 1) Main text, second paragraph. Johnson et al., 2013 is really derivative of the other earlier references, which should include Orzalli et al., 2012.Agreed; we referenced Orzalli et al., 2012, in the revised manuscript.2) Main text, second paragraph. IFI16 is associated with autoimmunity, but it not known how it "promotes" it.We revised the text to make this section more clear.3) Main text, sixth paragraph. This states that the chromatin inhibition hypothesis has not been tested directly. Orzalli et al., 2013 did compare gene expression of SV40 DNA in chromatin versus free DNA.Thank you; we revised the manuscript to include a brief statement on the in vivowork.Reviewer #2:[…] The proposed model of DNA sensing by 1D diffusion is interesting and novel. However, I have a number of concerns. In particular, the authors failed to show the importance of diffusion in filament assembly, which is the main conclusion of this manuscript. The length sensitivity observed between ~50 bp-200 bp in N, with N being the number of proteins that need to arrive simultaneously).The reviewer brings up an important point that is partly related to reviewer #1’s concerns about a possible cooperativity mechanism. We can exclude cluster formation taking place via three- dimensional search mechanisms in the absence of cooperativity: With our observation that a minimum number of four IFI16 molecules are needed to form a stable cluster and in the absence of cooperativity, chances that these four molecules will arrive at the same time on the same point of DNA are negligibly small . This is unlikely for the reasons that we described in our reply to Reviewer #1 (please see the discussion above).Another reason that makes me doubt about the importance of diffusion is the range of DNA length that IFI16 appears to sense. The authors observed the assembly rate increases with the DNA length but plateaus around ~200 bp. Since the authors clearly showed that IFI16 can diffuse far beyond 200 bp (>2-3 kb), this observation cannot explain the observed saturation at ~200 bp. On the other hand, this can be simply explained by the requirement of ~10 protomers to form a stable filament, as suggested by the authors. Note that there are proteins that form filaments in a nucleic acid length-dependent manner but without 1D diffusion. If diffusion is important for filament assembly, there should be a length dependence beyond 200 bp, especially at low protein concentration where the 3D diffusion-mediated filament assembly is significantly compromised. Again, without kinetic modeling that clearly shows the inconsistency with 3D diffusion and requirement for 1D diffusion, it is hard to arrive at the same conclusion as the authors.The reviewer brings up an important point. While it is true that a minimum of ~200 bp is needed to provide sufficient space to form a stable oligomer, it is not necessarily true that a further increase in length would further speed up the reaction kinetics. After all, lengthening DNA would also increase the average distance between neighboring proteins bound to DNA, and thus slow down their association via 1D diffusion [5]. We would like to point out two papers that we published in the last month addressing this particular issue. One of these papers [6] studies the association kinetics of two DNA-sliding entities and finds no strong DNA-length dependence. A theoretical paper [7] describes an analytical model based on a mean-field kinetic description of linear extended sinks (representing DNA) that can trap molecules present in a solution. The model presented there accurately describes the observed independence of reaction rate on DNA length and further demonstrates that the major contribution to association kinetics is one- dimensional sliding. While we agree with the reviewer that the data mentioned above alone cannot positively confirm the importance of a scanning component, it also cannot be used to exclude such a contribution.Another concern is that the 1D diffusion was shown only under flow. I understand that this is required to visualize diffusion (as you need to stretch the DNA), but this makes diffusion mostly uni-directional, and could likely affect the scanning processivity and the contribution of diffusion to filament assembly efficiency. More importantly, long DNA  collapses in the absence of flow, in which case 3D diffusion (or hopping), in comparison to 1D diffusion, could be more important for the filament assembly.We acquired the diffusion coefficients for individual IFI16 molecules and the HinAB construct on DNA templates, which were tethered at both ends to the surface and did not require a constant flow. Therefore, the robust 1D diffusion seen in Indeed, applying flow during the clustering experiments changes the diffusion behavior in terms of directionality and slightly in the value of the diffusion coefficient. We added a correction term for calculating the diffusion coefficient under flow , and also added a Monte-Carlo model, in which we simulated the clustering behavior with and without flow bias . Having now gained knowledge about the mechanism of IFI16 binding to DNA, i.e. its ability of 1D scanning over kbp lengths, we included this aspect better in our Discussion.Reviewer #3:1) Based on the FRET results shown in The footprint of IFI16 was determined using multiple dsDNA sizes and full-length protein [4]. Also as shown in the previous study by Morrone et al. [4], mutants with either the HinA or the HinB domain show a similar binding behavior on short DNA substrates (72 bp) as the full-length protein, albeit with a small reduction in DNA affinity in the absence of either domain. Due to the geometry of the Hin domains and the footprint of one Hin domain being ~ 7 bp [10], we do not expect a significant change in the footprint length.2) The authors propose that IFI16 scans along dsDNA  and 2. HWe agree that this aspect of Hin domain contribution to the binding is very interesting, especially in the context of a comparison with the DNA binding domain of Aim2 and its potential diffusion behavior. However, we feel that those experiments would be beyond the scope of this manuscript, which is focused on the distinction of self and foreign DNA.3) The authors show beautifully the movement of IFI16 along the lambda-phage dsDNA and propose that this diffusion is critical for IFI16-mediated immune responses. However, it is not clear if this movement is a property specifically required for triggering immune response rather than a property involved in the transcriptional regulatory functions of IFI16. Are the immobile clusters sufficient for triggering immune response? The authors could add to their Discussion to address these possibilities.We added this aspect to our conclusion and referenced the appropriate work suggesting PYD- domain dependent signaling.References1) Lu, A. et al. Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell 156, 1193-206 (2014).2) Vajjhala, P.R., Mirams, R.E. & Hill, J.M. Multiple binding sites on the pyrin domain of ASC protein allow self-association and interaction with NLRP3 protein. J Biol Chem 287, 41732-43 (2012).3) Orzalli, M.H., Conwell, S.E., Berrios, C., DeCaprio, J.A. & Knipe, D.M. Nuclear interferon-inducible protein 16 promotes silencing of herpesviral and transfected DNA. Proc Natl Acad Sci U S A 110, E4492-501 (2013).4) Morrone, S.R. et al. Cooperative assembly of IFI16 filaments on dsDNA provides insights into host defense strategy. Proc Natl Acad Sci USA 111, E62-71 (2014).5) Hu, T., Grosberg, A.Y. & Shklovskii, B.I. How proteins search for their specific sites on DNA: the role of DNA conformation. Biophys J 90, 2731-44 (2006).6) Turkin, A. et al. Speeding up biomolecular interactions by molecular sledding. Chemical Science (2016).7) Turkin, A., van Oijen, A.M. & Turkin, A.A. Theory of bimolecular reactions in a solution with linear traps: Application to the problem of target search on DNA. Physical Review E 92, 052703 (2015).8) Leith, J.S. et al. Sequence-dependent sliding kinetics of p53. Proc Natl Acad Sci U S A 109, 16552-7 (2012).9) Halford, S.E. & Marko, J.F. How do site-specific DNA-binding proteins find their targets? Nucleic Acids Res 32, 3040-52 (2004).10) Jin, T. et al. Structures of the HIN domain:DNA complexes reveal ligand binding and activation mechanisms of the AIM2 inflammasome and IFI16 receptor. Immunity 36, 561-71 (2012)


According to the gateway hypothesis, tobacco use is a gateway of cannabis use. However, there is increasing evidence that cannabis use also predicts the progression of tobacco use (reverse gateway hypothesis). Unfortunately, the importance of cannabis use compared to other predictors of tobacco use is less clear. The aim of this study was to examine which variables, in addition to cannabis use, best predict the onset of daily cigarette smoking in young men.n = 4,230).A total of 5,590 young Swiss men  provided data on their substance use, socio-demographic background, religion, health, social context, and personality at baseline and after 18 months. We modelled the predictors of progression to daily cigarette smoking using logistic regression analyses (In the multivariate overall model, use of cannabis remained among the strongest predictors for the onset of daily cigarette use. Daily cigarette use was also predicted by a lifetime use of at least 50 cigarettes, occasional cigarette use, educational level, religious affiliation, parental situation, peers with psychiatric problems, and sociability.Our results highlight the relevance of cannabis use compared to other potential predictors of the progression of tobacco use and thereby support the reverse gateway hypothesis. Many variables could be important for the progression of tobacco use. The identification of the relevant ones that best predict the progression of tobacco use is highly important because tobacco use is, by far, more widespread than cannabis and other illicit drug use and accounts for a significantly greater global burden of disease . IdentifOne of the discussed predictors of tobacco use is cannabis use. Tobacco use can act as a gateway to cannabis use , but theThe way of substance administration is probably among the most important connecting mechanisms of tobacco and cannabis use. In the qualitative study by Amos and colleagues , many paApart from the way of administration, the common liability model can also explain the strong association between tobacco and cannabis use. This model assumes that a common liability to using both licit and illicit drugs puts an individual at risk for using both legal and illegal substances, such as tobacco and cannabis. This liability may include a genetic and individual vulnerability, such as proneness to deviant personality and familial liability to addiction . Peer inOther potential predictors that could influence tobacco and/or cannabis use are religiosity and context variables such as socio-economic status or changes in social environments. Religious and pro-social activities are negatively associated with late-onset cannabis use  whereas The aim of this study was to explore how the onset of daily cigarette smoking can be best predicted from a comprehensive set of variables, including cannabis use.The present data are part of the Cohort Study on Substance Use Risk Factors (C-SURF), a longitudinal study designed to assess substance use patterns and their related consequences in young Swiss men. Enrolment in the study occurred between 2010 and 2011 in three Swiss army recruitment centres, which cover 21 of the 26 Swiss cantons. (A canton is a type of administrative division of a country and the Swiss cantons are semi-sovereign states.) Switzerland has a mandatory army recruitment process: virtually all young men are contacted at approximately 19 years of age for determination of their eligibility for military or civil service. Thus, not only individuals who were finally selected to serve in the army were enrolled in the study, but a virtually complete census of the Swiss male population in this age group was eligible. The participants filled in the questionnaire online or via mail and were rewarded with a voucher of 30 Swiss Francs (CHF).The follow-up assessment was conducted approximately 15 months after the baseline measurement, and the participants were reimbursed with a similar voucher. The participants who filled in both questionnaires received an additional voucher (30 CHF). Between the assessments, the participants were invited twice to update their contact details online. Each of these two updates was rewarded with a voucher (5 CHF) once the second questionnaire was completed.The Ethics Committee for Clinical Research of Lausanne University Medical School approved the study (Protocol No. 15/07).A total of 15,074 young men visited the recruitment centres. Among them, 1,829 (12.1 %) did not meet the research staff because they were sick , were randomly selected to participate in another study , or wereFor the model of progression from no or occasional cigarette use at baseline to daily cigarette use at follow-up, we excluded 1,275 of the 5,990 individuals (21.3 %) because of daily cigarette use already at baseline and an additional 485 individuals (8.1 %) because of missing data, resulting in a final sample of 4,230 individuals.The participants indicating cigarette use during the previous 12 months were asked how often they usually smoke cigarettes. The possible answers  were dichotomised (daily vs. non-daily use). For the analysis of the onset of daily cigarette smoking, the sample included all the participants who used cigarettes less than daily or not at all at baseline. Among these participants, reporting daily cigarette smoking at follow-up was classified as the onset of cigarette use.All the predictor variables were measured at baseline.The socio-demographic predictors included age in years and the highest completed level of education divided into two categories: a lower educational level  and a higher educational level . Additional socio-demographic predictors included the housing situation , the means of subsistence , living in a partnership (yes/no), and the number of siblings.Religious denomination was assessed by the question “What is your religion (even if you do not practice or believe in God)?” with nine response categories, which we merged into four categories: Christian religion, Muslim religion, other religion, and no religion. To measure religiosity, we used the first question of the Religious Background and Behaviour Questionnaire (RBB)  with thePhysical and mental health were measured by the Physical Component Summary and the Mental Component Summary of the 12-Item Short-Form Health Survey (SF-12) , the MajThe parental situation was assessed by a question derived from the Alcohol Use Disorder and Associated Disabilities Interview Schedule-IV (AUDADIS-IV) . The parParenting was assessed by three variables used in the ESPAD. Two questions  your mother and (b) your father?”) measured the participants’ retrospective satisfaction with the relationship with their parents on scales ranging from 1 (“very satisfied”) to 5 . The scores were dichotomised at the median of the mean of the items assessing maternal and paternal relationships. Two questions with response scales ranging from 1  to 5  were used to derive parental regulation at the age of 15 years: “My parents set definite rules about what I was allowed to do (a) at home and (b) outside the home”. The scores were averaged and dichotomised at the median. The retrospective assessment of parental knowledge of peers and the whereabouts at age 15 were derived by averaging and dichotomising the scores obtained from the responses to two five-point items: “My parents knew (a) whom I was with and (b) where I was in the evenings.” Parental rule setting and knowledge of peers and the whereabouts was asked at around age 15 because this is the time when peer influences become stronger, and particularly meeting with friends without the participation of parent increases . For exaThe lifetime prevalence of psychiatric, alcohol or drug problems in the parents was assessed separately for both parents. The participants indicated whether a significant problem had ever been present in one or several domains . A similar question addressed previous significant alcohol, drug, or psychiatric problems in peers. Peer pressure was assessed by a shortened version of the Peer Pressure Inventory (PPI) .Lifetime use of alcohol was assessed by the question “Did you have at least 12 alcoholic standard drinks in your entire life?” Examples for alcoholic standard drinks were pictured. Furthermore, the age of the first use of at least one standard alcoholic drink, the 12-month prevalence, and at-risk drinking were assessed. The possible answers  were dichotomised (daily vs. non-daily use). Participants who indicated ‘yes’ were classified by the Alcohol Use Disorders Identification Test (AUDIT-C)  as not aTo assess the lifetime use of cigarettes, participants indicated whether they consumed at least 50 cigarettes in their life. Furthermore, the age of first cigarette smoking and the 12-month prevalence of cigarette smoking were assessed (see above). Additionally, the 12-month prevalence for the use of tobacco products other than cigarettes , snus, snuff, chewing tobacco, cigars/cigarillos, tobacco pipes) was measured.The lifetime use of cannabis was assessed by asking “Have you ever consumed cannabis , more than just to try?” Subsequent questions measured the age of first cannabis use and problematic cannabis use, which was assessed with the Cannabis Use Disorders Identification Test (CUDIT) . AlthougThe lifetime use of illicit drugs other than cannabis at baseline was assessed by a series of questions measuring the frequency of use of 15 illicit drugs within the course of the individual’s life . The lifetime use of illicit drugs was defined as having used at least one of these substances at least once.Screening for adult attention deficit syndrome was performed with the Attention Deficit Syndrome Self Report Scale (ASRS-v1.1) . SensatiR2 was calculated as a goodness-of-fit measure for all multivariate models. All the analyses were performed using SPSS version 20 [p < 0.05 was set as the significance level.Starting with separate logistic regression analyses , we evaluated the potential of each baseline variable to predict the onset of daily cigarette use. To reduce multicollinearity within the final multivariate model, we developed separate multivariate prediction models for each of the following categories of predictor variables: (1) socio-demographics, (2) religion and spirituality, (3) health and health behaviour, (4) social context, (5) substance use, and (6) personality. Variable selection comprised the following steps: (1) Significant predictors from the univariate analyses were entered into the separate models. (2) Variables that were not significant were removed manually one by one; variables with the highest p-values were removed first (backward selection). (3) To account for suppressor effects, the resulting models were verified by tentatively adding the excluded variables separately. Only significant variables were retained in the category-specific multivariate models (forward selection). Based on the results of these models, we developed one final model for the onset of daily cigarette use. Variable selection was conducted in an analogous way as described above, with the exception of including all significant predictors from the category-specific models at step (1). Nagelkerke’s rsion 20 , and p <The baseline characteristics of the 4,230 participants included in the analysis of the onset of daily cigarette smoking are displayed in Table Between baseline and follow-up, 269 (6.4 %) participants progressed to daily cigarette smoking. Table This study aimed to examine the role of cannabis use in the progression of tobacco use, as previously investigated in several studies dedicated to the reverse gateway hypothesis –5, 47. CIn our study, the factors referring to a genetic vulnerability such as a psychiatric disorder of the father/mother or externalizing and/or delinquent personality traits  did not remain significant in the overall predictor model. This is in line with the conclusion of Ramo and colleagues  who founAmong the religion and spirituality factors, having no religion was a relevant predictor for the onset of daily cigarette smoking. Whether substantial gains or losses in religiosity from childhood to adulthood occurred, a predictor that has been reported to be associated with substance use and misuse in the general U.S. population , was notConsidering the amount of variance explained by the category-specific models, substance abuse explained the major part of variance (25.1 %) compared to the other categories (0.6 % to 4.6 %).One limitation of this study is that the participants were only observed for a relatively short time period because they were reassessed at one time point after 15 months. In addition, there was a period of approximately three months that was not included in the assessments because the follow-up assessment only examined the preceding 12 months. In order to analyse potential moderators and mediators in the reverse gateway scenario, cohort studies including multiple assessments over a longer time period are required. A further limitation is the dichotomized outcome variable (daily cigarette smoking vs. non-daily cigarette smoking). This implicates that a group of 216 smokers who smoked cigarettes on 5 to 6 days a week already at baseline were treated as non-daily smokers and therefore includes in the analysis. These occasional smokers may have had a particularly high chance to proceed to daily cigarette smoking and to be treated as participants with “onset of daily cigarette smoking” which may seem an artificial transition, given they already smoked already nearly daily at baseline.According to our findings, appropriate interventions to prevent young adults from daily cigarette use should specifically address occasional cigarette smokers and users of cannabis that mix and smoke these substances together.


The mechanisms involved in the etiology of adverse drug reactions (ADRs) are complex. A small proportion of ADRs are due to drug hypersensitivity where the immune response causes an unexpected and severe clinical reaction. The mechanisms of drug hypersensitivity are not yet fully understood, but genome wide screens have identified an association with HLA alleles and certain drugs. Furthermore, functional studies have identified drug-responsive T-cells in blood and tissues of hypersensitive patients. Thus, the frequency and severity of a reaction is a function of the chemistry of the drug, the biology of the immune cell and the genotype of the individual.We sought to establish a biobank of lymphocytes isolated from HLA-typed volunteers to investigate how drugs and their metabolites become antigens and interact with the HLA molecules and T cells to generate an immune response. We have recruited 900 volunteers so far and 400 of these have been HLA typed.The typed cohort is representative of the North-West population and consists of 64% female and 36% male volunteers with a mean age of 29 years . The ethnicity is primarily Caucasian (84%); however, a special effort was made to include other ethnicities  in order to increase the HLA allelic diversity within the cohort. All major Caucasian haplotypes are represented. Using a DC priming assay we have investigated whether we could prime naïve T cells to various drugs. Nitroso sulfamethoxazole was included in all assays as a positive control and drug-specific proliferative responses were generated from 35 out of 38 donors. Strong responses were also generated to carbamazepine in two HLA-B*1502 donors (n=2), to flucloxacillin in one HLA-B*5701 donor (n=5), to pipericillin in 5 donors (n=10) and to Bandrowski's base in all donors (n=7). In contrast, no positive responses were detected to ximelagatran in four HLA-DRB1*0701 donors and to lumiracoxib in four HLA-DRB1*1501 donors. However, drug-specific T cell clones could still be isolated from 3 donors when only weak responses to flucloxacillin were detected.This work shows that drug specific responses can be generated from naïve T cells and suggests that not all hypersensitivity reactions are due to cross-reactivity with existing memory cells.


Members of the CD44 family of transmembrane glycoproteins emerge as major signal transduction control units. CD44 isoforms participate in several signaling pathways ranging from growth factor-induced signaling to Wnt-regulated pathways. The role of the CD44 family members in tumor progression and metastasis is most likely linked to the function of the various isoforms as signaling hubs. Increasing evidence suggests that these proteins are not solely cancer stem cell (CSC) markers but are directly involved in tumor and metastasis initiation. It is foreseeable that a link between the expression of CD44 isoforms in CSCs and their function as signaling regulators will be drawn in a near future. The term CD44 designates a large family of transmembrane glycoproteins belonging to the class of cell adhesion molecules. CD44 family members are involved in physiological processes such as hematopoiesis, lymphocyte homing, or limb development [reviewed in Ref. ]. All CDCD44s is expressed in nearly all tissues whereas the expression of CD44v isoforms is restricted to specific cell types . This wA participation of various CD44 isoforms in the activation of other RTKs  was reviewed in Ref. . For exaA completely different receptor, namely, CXCR4 also appears to be dependent of CD44 . This tiXenopus laevis embryos impaired expression of Wnt-regulated genes such as eng-2 and tcf-4. CD44 is therefore able to act as a Wnt-target gene as well as a Wnt regulator.More recently, the Wnt-target gene CD44  was showSince CD44s and CD44v isoforms are involved in tumor progression and metastasis alternative splicing of CD44 seems to be a decisive event controlling the progression of cancer. Several years ago, a mini-gene construct was used to investigate exon v5 alternative splicing and the relevance of signal transduction for this process was shown . The nuc+ phenotype . EMT is a process by which epithelial cells loose their polarity, gain invasive properties, and acquire mesenchymal features. On one hand, EMT was shown to induce a CD44henotype . On the henotype . hnRNPM-+-independent cystine transporter xCT and suppressed lung colonization.In contrast to the above described studies, colonization of the lung by 4T1 mouse breast cancer cells was shown to be dependent on the switch from CD44s to CD44v isoforms . This tiIn the same line, the activation of the Wnt pathway, a central player in EMT, induced CD44v6 expression in colorectal cancer cells [reviewed in Ref. ] as well+. CD44 is also expressed on several other types of CSCs including pancreatic and colorectal CSCs . A mAb  of LSCs . Taken t of LSCs  might beFor long, the role of CD44 in tumor progression and metastasis remained unclear. Many lines of evidence indicate that CD44 organizes a signaling platform at the cell surface by acting as a co-receptor for various types of cell surface receptors Figure . In addiThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Incorrect lead placement may result in unnecessary therapeutic interventions. We present a case report of 53-year-old man with new inferior T-wave inversions in the 12-lead electrocardiogram (ECG) noted during routine followup of hypertension without any cardiovascular symptoms. The common right arm/left arm lead switch is easily recognizable, but many other lead placement errors are possible. The less common ones are often difficult to detect, but errors are quite important clinically, as they may induce erroneous diagnoses . The proA fifty-tree-year old man with new T-wave inversions in the 12-lead ECG noted during a routine followup for hypertension. The patient also had dyslipidemia, but no history of coronary disease or diabetes. His hypertension was well controlled on a combination of beta-blocker and calcium-blocker; in addition, he used a statin.The physical examination was entirely normal. The ECG obtained sinus rhythm at 54 beats per minute, with rS configuration in lead I and aVL, indicating a left posterior fascicular block and narrow Q waves in III and aVF accompanied by deeply inverted T waves in both leads. In the precordial leads, we observed an abrupt transition in V2 with a qR complex and inverted T wave . InitialBefore cardiological checkup, it was decided to perform a new ECG  that proInterchange between electrodes of the left arm and the left leg in a patient with left anterior fascicular block in the basal ECG may raise the suspicion of inferior myocardial infarction with inverted T waves in leads III and aVF , 3. CrosIn our case, there was an abnormal transition in precordial leads with a qR complex morphology in V2 that did not concur with either V1 or V3. If there is a marked discrepancy in the ECG pattern among leads that reflect electrical activity in a similar anatomical location, for example, if lead I and aVL QRS complex is different from that of V5 and V6, lead reversal should be suspected [The ECG morphology registered in the frontal plane in this case can be considered as a mixture of frontal and horizontal planes because of interchange between electrodes of the left arm and V2 . Incorrect lead placement may include application of the electrode in a wrong location, connection of the cable to the wrong electrode, or both . The exp


Nigeria’s high perinatal mortality rate (PNMR) could be most effectively reduced by targeting factors that are associated with increased newborn deaths. Low access to skilled birth attendants (SBAs) and weak health system are recognized factors associated with high PNMR but other socio-demographic and reproductive factors could have significant influences as well. Identification of the major factors associated with high PNMR would be required in designing interventions to improve perinatal outcomes.For this cross-sectional study, data from the Nigeria Demographic and Health Survey 2008 were used to estimate the PNMR of non-hospital births in identified socio-demographic and reproductive situations that are known to influence PNMR. The estimated PNMR were compared using logistic regression analysis.The PNMR was 36 per 1000 live births. North central region had the lowest PNMR while the south east region had the highest rate . Other correlates of high PNMR were belonging to the poorest wealth quintile , maternal age group 15–19 years , multiple birth , history of previous perinatal death , birth interval shorter than 18 months  and having a small birth size . Birth attendant, place of birth, parity, maternal education and rural/urban residence had no association with PNMR.Reproductive factors that require midwifery skills were found to contribute most to PNMR. We recommend general strengthening of the health system, recruitment of SBAs and retraining of available birth attendants with emphasis on identification and referral of complicated cases. Family planning should be a core MCH activity to address the issues of teenage pregnancy and short pregnancy intervals. It would be almost impossible to achieve the Millennium Development Goal 4 (MDG-4) at the current rate of decline of annual under-5 deaths from 10.5 million in 1990 to 6.6 million in 2012 . IntrapaIn Nigeria, there is an acute shortage of midwives and obstetricians, and a virtual unavailability of these cadres of health workers at the primary health care (PHC) level . MoreoveWith a population of over 140 million and perinatal mortality rate (PNMR) of 39 per 1000 live births, , Nigeriaa priori socio-demographic and reproductive characteristics that are known to influence the PNMR. The result of the study is expected to provide evidence that would guide policies on perinatal health and also identify directions for further studies on perinatal health.Recent interventions by the federal government of Nigeria to improve pregnancy outcomes mainly target increasing the number of childbirths at the PHC clinics . Media cThe study was based on an analysis of data from the Nigeria Demographic and Health Survey 2008 (Nigeria DHS 2008) which took place from June to October 2008 .The NigeThe main outcome measure for this study was the perinatal mortality rate. This was estimated from early neonatal deaths of births from 2003–2008; and stillbirths (pregnancies that lasted for 28 weeks or more but did not result in live birth) from 2003–2008. Early neonatal deaths and stillbirths were in turn respectively derived using the variables for year of birth ( b2) and age at death (b6 ) for early neonatal death, and year of non-viable pregnancy (v230 ) and its duration before it terminated ( v233) for stillbirth.a priori exposure variables that are known or thought to affect perinatal mortality include the following:Birth attendant was the main exposure variable. Other Demographic factors: region, residence , wealth index, mother’s age and mother’s education.Reproductive factors: mother’s parity, previous mortality experience, place of delivery, number of babies (singleton or multiple) length of the birth intervals and size of baby at birth.Births at PHC centers, health posts, other non-hospital public and private places, respondents’ homes and other homes were included in the main analyses. Supplementary estimation of the PNMR of hospital births  was done for the purpose of comparison with the PNMR of non-hospital births where appropriate. We reported the PNMR of hospital births only in those circumstances where the pattern of perinatal death in hospital births was different from that of non-hospital births.R was already cleaned and recoded. Missing dates were not allowed as dates were calculated and imputed for them. Missing values, inconsistent and impossible values and “I don’t know” responses were assigned special value. Such values were identified and recoded as missing values for purpose of the current analysis. In order to answer the research question, we generated a number of new variables from existing variables and recoded some variables.The dataset obtained online from Measure EvaluationR statistical package version 12. Descriptive and logistic analyses were used to estimate and compare the PNMR across identified demographic and reproductive characteristics. Observed differences were considered significant at the p value of <0.05; 95% confidence interval. The “gen weight” and “svyset” command functions of the stata statistical software were used to account for the complex survey features of the HDS dataset.The data analysis was done with StataThe ethical considerations and approval for the collection of the primary data has been described . PermissThe overall response rate of the DHS 2008 was 94.9%. Out of a total of 32, 394 births, 25, 817 (79.7%) were non-hospital births while 6,577 (20.3%) were hospital births. The national perinatal mortality rate (PNMR) of non-hospital births in Nigeria was 36 per 1000 live births (95% CI: 33.4-39.3). This is lower than the PNMR of 46 per 1000 live births for hospital births (95% CI: 40–53.3).The PNMR was lowest in the north central region 27/1000) and highest in the south east region (47/1000). The rate was 37/1000 in the rural areas and 35/1000 in the urban areas. Those in the richer wealth quintile had the lowest PNMR of 28/1000 while those in the richest quintiles had the highest rate (47/1000). However among hospital births, the PNMR showed a different pattern as follows: 76.6/1000  for poorest, 52.4/1000  for poorer, 55.2/1000  for middle, 44.2/1000  for richer, and 40.2/1000  for the richest, p = 0.007. Maternal age group 25–29 years was associated with the lowest PNMR (33/1000) while the age group 15–19 years was associated with the highest rate (50/1000). The PNMR increased with increasing level of maternal formal education with higher education being associated with the highest rate. For hospital births, the PNMR showed an inverse relationship with the level of mother’s education thus: 64/1000, 53/1000, 41.4/1000 and 38/1000 respectively for no maternal education, primary education, secondary education and tertiary education (P = 0.0736). The distribution of perinatal mortality in non-hospital births according to socio-demographic factors is shown in Table 000 and hWith a PNMR of 33/1000, parity 2–4 was associated with the lowest perinatal deaths while primiparity was associated with the highest rate (39/1000). Those with a history of previous mortality experience were at a much higher risk of perinatal death than those without such history (79/1000 versus 28/1000). Delivery at private frontline health facility was associated with lower perinatal deaths than delivery at public PHC facility. The perinatal mortality associated with multiple births was 121/1000 compared to 34/1000 for singleton births. At a PNMR of 28/1000, birth interval of 25 months and above was associated with much lower perinatal deaths than the shorter birth intervals while an interval of less than 18 months was associated with the highest perinatal mortality. Births assisted by CHEWs were associated with a PNMR of 31/1000 compared to 41/1000 for those assisted by nurses. Being born small was associated with a PNMR of 75/1000 while having “average” birth size was associated with a PNMR of 30/1000. Table Table The PNMR among non-hospital births in Nigeria (2003–2008) was 36 per 1000 live births while the PNMR of hospital births was 46 per 1000 live births.After adjusting for known confounding factors using multiple regression analysis, there was no significant difference in perinatal mortality according to rural versus urban residence, birth assistant and place of birth. Reproductive characteristics such as teenage pregnancy, short birth interval, multiple gestation, bad obstetric history and small birth size were found to be the major contributors to the PNMR among non-hospital births in Nigeria.The main findings of the study were:The PNMR was 36/1000 live births in births that occurred at the primary health care facilities and homes. This was lower than the PNMR of 46/1000 live births when only hospital births were analyzed and also lower than the PNMR of 39/1000 live births when hospital births and non-hospital births were combined  (result a.The PNMR was highest in the south east region (47/1000) and lowest in north central region (27/1000). The relatively higher PNMR in the south east region was a departure from previous DHS patterns where the region shared the best reproductive and child health indicators with the south west region . The resPerinatal mortality was higher in the rural than the urban areas (37/1000 versus 35/1000). This is consistent with previous Nigeria DHS surveys and the global pattern , 32, 33.People in the richer and middle wealth quintiles had lower PNMR than the poorer and the poorest. It is noteworthy that the very high PNMR initially observed in association with the richest wealth status on descriptive analysis disappeared following multiple adjustment for confounding factors suggesting that some factors other than high economic status were responsible for the observed association. Note also that for hospital births, the PNMR was consistently highest in the poorest socio-economic status and lowest in the richest wealth group. Because government-owned hospitals and private hospitals in Nigeria charge service fees, the rich are more likely than the poor to choose hospitals for prenatal and perinatal care. On the other hand, a larger proportion of the poor than the rich who reported hospital birth might have been referred to hospital from homes and from PHC clinics as complicated cases which have poorer prognosis.As in other studies , 28, extParity group 2–4 was found to be associated with the lowest PNMR. Other studies have also identified primiparity and grand-multiparity as risk factors for perinatal death , 35. ComMultiple birth was found to be associated with over threefold increase in perinatal mortality compared with singleton births . The perinatal outcome of multiple pregnancies is dependent on the quality of care during pregnancy, childbirth and postpartum period. With standard quality of care, PNMR of twin births do not differ significantly from singleton births . Unlike Birth interval of greater than two years was found to be associated with the best perinatal outcome; those born less than 18 months after a previous birth fared the worst . Short birth interval is a recognized risk factor for perinatal death [Perinatal mortality rate in birth assisted by the CHEWs was 31/1000 compared with 41/1000 for those assisted by nurses Table . The assAverage birth size was associated with the lowest PNMR while small birth size had the greatest risk of perinatal death. Low birth weight as a result of prematurity or intrauterine growth restriction is a recognized risk factor for perinatal death .a priori adverse influences of the reproductive factors on perinatal mortality were exaggerated. The negative effects of such reproductive factors as multiple births and previous mortality experiences could be effectively minimized by early identification and referral of such cases to hospitals as the PHC clinics are disallowed to manage complicated pregnancies such as these. Teenage pregnancies and short pregnancy intervals are best prevented through appropriate family planning interventions. Unfortunately, the weak health system and poor clinical skills  of birth attendants permitted these factors to exert their maximum negative influences on perinatal mortality. When the high PNMR (36/1000) of non-hospital births is considered together with the even higher PNMR (46/1000) of hospital births, the picture becomes clearer that of a health system that is too weak to respond to the basic demands of safe perinatal care. Many of the referred cases reach the hospitals too late to be rescued and some of the hospitals are not adequately equipped to manage the referred cases.In summary, the findings of this study suggest that women who give birth at home or at PHC facilities in Nigeria have some common underlying residual socio-demographic risk factors for poor perinatal outcomes that overshadow the positive influences of wealth and education. Cultural practices including religious practices could exert such a homogenizing influence. Further studies are required to uncover such factors. On the other hand, the The factors that contribute most to perinatal death in non-hospital births in Nigeria include teenage pregnancy, short pregnancy interval, multiple gestations, previous perinatal deaths and small size at birth. The myriads of adverse pregnancy outcomes resulting from teenage pregnancy and short pregnancy interval are effectively preventable by family planning. Multiple gestations and the history of previous perinatal deaths are cases for hospital care that should not be managed at homes or at the PHC clinics. The absence of improved PNMR in PHC facility births when compared with home births could be due to lack of SBAs at the PHC clinics, referral bias or because high-risk pregnant women preferentially selected to have childbirth at PHC facilities rather than at home. However, the consistently high PNMR at all locations of birth including hospitals suggests a health system that is generally weak.We recommend general strengthening of the health system including provision of life-saving equipment, medicines and consumables, recruitment of trained SBAs and adequate competency-based retraining of existing birth attendants as well as establishment of functional referral and transportation systems before mobilizing the public for increased uptake of PHC facility-based perinatal services. The emphases of SBA training and retraining should include the skills to identify complicated pregnancies and labors that must be referred to hospital without delay. Referral hospitals should be equipped with requisite life-saving equipment and skilled personnel to competently manage complicated cases referred from homes and PHC clinics. We further recommend that family planning should henceforth be made one of the core MCH activities because of its numerous benefits for perinatal health. Finally, public awareness should be created on the benefits of family planning and on the dangers of managing high-risk pregnancies and labors at home or at the PHC clinics. We believe that these recommendations would also benefit other countries with similar weak health systems as Nigeria.Nigeria DHS 2008 was a nationally representative survey with large sample size and associated high power and precision to detect differences in the indicators of interest including perinatal mortality. The response rate of 94.9% was high for such a large population survey. To ensure accuracy and uniformity of information collected, the study questionnaire was translated to local languages and back-translated to English, contained consistency check questions, and were administered by trained field officers with multiple levels of supervision. Missing values were minimal. The study was therefore based on robust data base. Although the DHS 2008 was a cross-sectional survey, the periodic repeats of the DHS have made it useful for tracking progress in indicators of interest.The study was based on respondents’ recall of previous events which is prone to recall bias. The error in remembering the age at death involving very young ages is a recognized problem in DHS and other surveys . MoreoveIt is intuitive that women with higher-risk pregnancies are more likely than their lower-risk counterparts to have health facility childbirths by choice or through referral. This could have lead to worse perinatal outcomes in the PHC facility births compared to home births. The DHS dataset did not contain the information needed to adequately control for these potential risk-related biases.There were probably some misclassification of birth assistants by the respondents as the midwives, nurses and CHEWs wear similar uniforms and perform similar duties at the PHC facilities. Because the generic job title of female health workers in most rural communities in Nigeria is “nurse”, some CHEWs might have been misclassified as nurses. Misclassification in the reverse direction is possible but less likely.These limitations are acknowledged.aThe 2013 Nigeria DHS showed that the south east region had the second lowest PNMR at 36/1000.


Aims and method Electronic patient records were used to investigate the level of engagement and treatment that patients with very late-onset schizophrenia-like psychosis (VLOSLP) had with mental health services.Results Of 131 patients assessed and diagnosed, 63 (48%) were taking antipsychotic treatment at 3 months, 46 (35%) at 6 months and 36 (27%) at 12 months. At discharge from mental health services, 54% of patients had failed to engage with services or became lost to follow-up, 18% had engaged with services but were not taking antipsychotic medication and only 28% were taking treatment.Clinical implications Results showed that less than half of the patients with VLOSLP were commenced on antipsychotic treatment and less than a third remained on treatment at 1 year or at point of discharge. This highlights the need for services to consider being more assertive in taking potentially effective treatment to this patient group. Little is known about how many patients seen within specialist mental health services are engaged by those services and given antipsychotic treatment. To address this, we conducted a retrospective electronic patient record search.A Cochrane review6 in contact with services between 1 January 2007 and 21 August 2014. All identified patients' notes were reviewed and those who scored 24 or less on the standardised mini-mental state examination7 or had a concurrent diagnosis of an organic mental disorder (ICD-10 F00–F09)6 were excluded. Other exclusion criteria included any evidence that psychosis onset had been before the age of 60, insufficient recorded information to confirm diagnosis and less than 1 year of follow-up completed at time of CRIS search.We used the Clinical Record Interactive Search (CRIS) system, developed within the National Institute for Health Research (NIHR) Mental Health Biomedical Research Centre, South London and Maudsley NHS Foundation Trust, to extract the records of all patients aged 60 years and above with a diagnosis of schizophrenia, schizotypal and/or delusional disorders (ICD-10 F20–F29)The first face-to-face contact of mental health services with a patient was regarded as the start point of each service episode. Records were reviewed at 3 months to assess initial engagement with services. If a patient was discharged before 3 months, the last documented episode was used as an alternative. Two further engagement points, at 6 months and 12 months, were also examined. The length of each episode was also extracted and the level of engagement at the point of discharge from services was assessed.Ethics approval for CRIS was given by the National Research Ethics Service Oxford REC C.n = 45), cognitive impairment (n = 216), onset of psychosis before age 60 years (n = 150), insufficient information to confirm diagnosis (n = 32) or in contact with services for less than 12 months preceding the search date (n = 61). Of the remaining 131 patients, 84 (64.1%) were female, 40 (30.5%) were White British, and 35 (26.7%) were living with a partner or family member. The extent of engagement with services and antipsychotic treatment at 3, 6 and 12 months is shown in Search of the CRIS system initially identified 635 patients. Following inspection of the individual records, patients were excluded because of incorrect coding (2 The illness is viewed as a functional psychosis with symptoms that will respond to antipsychotic drugs.5 Our data show that less than half of patients with VLOSLP were commenced on antipsychotic treatment and less than a third remained on treatment at 1 year or at the point of discharge from services. This is a surprising, even disappointing, result. An important barrier to acceptance of antipsychotic treatment by patients with VLOSLP is the low level of insight into presence of mental health difficulties or need for treatment.8 Although treatment-related decision-making capacity has not been specifically investigated in VLOSLP, studies of middle-aged and older patients with schizophrenia have suggested that cognitive test scores, rather than psychopathology ratings, associate most strongly with the understanding and reasoning components of capacity.9 Patients with VLOSLP do not, however, have demonstrably abnormal brain imaging10 and do not inevitably progress to develop dementia,12 so cognitive impairment is unlikely to explain their inability to evaluate their illness or the need for treatment. Reluctance by clinicians to use mental health law to deliver compulsory treatment when patients will not accept the case for antipsychotic treatment is indicated by the small percentage of cases where this happened. Although there are limited studies to compare this with, it is in keeping with the lower rates of patients on longer-term detention within the above-65 population.13 This may reflect a view that elderly patients with psychosis are somehow less ‘risky’ and that a failure to enforce treatment is therefore justifiable. In fact, untreated symptoms of VLOSLP are frightening and disabling, lead patients to place themselves at significant risk and damage relationships with family and neighbours. Comparably low levels of psychosis treatment in younger people would be completely unacceptable and specialist mental health services for older people should actively consider whether they should be more assertive in taking potentially effective treatment to this vulnerable patient group.The diagnostic concept of VLOSLP emerged from an international consensus meeting held to advance research on a patient group with first onset of delusions and/or hallucinations after the age of 60 years, in the absence of affective disorder or demonstrable brain disease such as dementia.


Previous psychophysical studies have reported conflicting results concerning the effects of short-term visual deprivation upon tactile acuity. Some studies have found that 45 to 90 minutes of total light deprivation produce significant improvements in participants' tactile acuity as measured with a grating orientation discrimination task. In contrast, a single 2011 study found no such improvement while attempting to replicate these earlier findings. A primary goal of the current experiment was to resolve this discrepancy in the literature by evaluating the effects of a 90-minute period of total light deprivation upon tactile grating orientation discrimination. We also evaluated the potential effect of short-term deprivation upon haptic 3-D shape discrimination using a set of naturally-shaped solid objects. According to previous research, short-term deprivation enhances performance in a tactile 2-D shape discrimination task – perhaps a similar improvement also occurs for haptic 3-D shape discrimination. The results of the current investigation demonstrate that not only does short-term visual deprivation not enhance tactile acuity, it additionally has no effect upon haptic 3-D shape discrimination. While visual deprivation had no effect in our study, there was a significant effect of experience and learning for the grating orientation task – the participants' tactile acuity improved over time, independent of whether they had, or had not, experienced visual deprivation. By the 1880's, it was well known from experiments performed by David Ferrier per se, Merabet et al. Many studies have now found that blindness is associated with enhancements in behavioral performance for non-visual sensory and perceptual tasks Evidence now exists to indicate that performance for some tactile tasks can be facilitated by only a few hours of visual deprivation. For example, Weisser et al. The experiment was approved by the Western Kentucky University Human Subjects Review Board. The participants were students at Western Kentucky University, and all participants gave written consent prior to participation in the experiment.2 containing furniture, tables, plants, etc). The blindfold eliminated all light when it was worn and was similar to those used in previous studies Twenty-eight younger adults participated in the experiment and were randomly divided into two groups. Fourteen participants  were visually deprived  throughout a large portion of the experiment, while 14 additional participants  were not visually deprived  and could see the entirety of our laboratory  or an Apple PowerMacintosh G4 computer (for the haptic solid shape discrimination task). The participants' judgments were entered into the computers for later analysis.Capsicum annuum, that have been regularly used in previous investigations The experimental stimuli for the tactile acuity task were tactile gratings , used previously by both our laboratory and others The tactile acuity of all participants was assessed at the beginning of the experiment. Following this, half of the participants were randomly assigned to the visual deprivation condition; these participants were then blindfolded (they remained blindfolded throughout the entire remainder of the experiment). After a 90-minute delay , all participants' tactile acuity was measured again. Following the second evaluation of tactile acuity, the participants' ability to discriminate solid shape was assessed. After completing the second evaluation of their tactile acuity and the shape discrimination task, the visually deprived participants were finally (after about 2.5 to 3 hours) allowed to remove their blindfold. No participant in the visually deprived condition ever reported detecting any light whatsoever. Nevertheless, we felt that it was important to demonstrate and ensure the integrity of the blindfold. Throughout the experiment  we repeatedly tested each participant to ensure total light deprivation. The room light (a 75-watt incandescent lamp) was turned on and off according to a random schedule for at least ten trials on each occasion; the participants were asked to judge on each trial whether the room lights were on or off. The participants' detection performance was no better than chance at any of the three time periods , demonstrating that the blindfold was effective in eliminating light.The procedures used for determining the participants' grating orientation thresholds  were similar to those used both by ourselves same shape or had different shapes. The participants were required to judge whether each pair of objects were the same or different. Only the most difficult pairs of objects to distinguish from our previous investigations were used: Objects 1 and 3, Objects 1 and 7, Objects 2 and 11, Objects 3 and 7, Objects 3 and 8, and Objects 5 and 12. Each participant judged a total of 96 pairs of objects . Within each block, half of the trials were devoted to the different object pairs, while the same object pairs were presented on the remaining trials . The order of same versus different trials was randomly determined within a block; however, each different pair was presented twice within a block. For each different pair, the order of presentation was randomized .The procedures used for the solid shape discrimination task were similar to those used by Norman et al. The participants' tactile acuities are plotted in x  = 200, p = .91, 2-tailed). A power analysis The results for the solid shape haptic discrimination task are shown in Prior research by four different laboratories, including our own not visually deprived. It is thus likely that their participants' tactile acuity improved during the second measurement session as a result of practice Click here for additional data file.Dataset S2Individual participant tactile acuities. Each participant's tactile acuity was assessed using a grating orientation discrimination task.(XLS)Click here for additional data file.Dataset S3Individual participant shape discrimination performance.(XLS)Click here for additional data file.


Medication non-adherence is a prevalent problem worldwide but up to today, no gold standard is available to assess such behavior. This study was to evaluate the psychometric properties, particularly the concurrent validity of the English version of the Malaysian Medication Adherence Scale (MALMAS) among people with type 2 diabetes in Malaysia. Individuals with type 2 diabetes, aged 21 years and above, using at least one anti-diabetes agent and could communicate in English were recruited. The MALMAS was compared with the 8-item Morisky Medication Adherence Scale (MMAS-8) to assess its convergent validity while concurrent validity was evaluated based on the levels of glycated hemoglobin (HbA1C). Participants answered the MALMAS twice: at baseline and 4 weeks later. The study involved 136 participants. The MALMAS achieved acceptable internal consistency  and stable reliability as the test-retest scores showed fair correlation (Spearman’s rho=0.412). The MALMAS has good correlation with the MMAS-8 (Spearman’s rho=0.715). Participants who were adherent to their anti-diabetes medications had significantly lower median HbA1C values than those who were non-adherence . The odds of participants who were adherent to their medications achieving good glycemic control was 3.36 times  of those who were non-adherence. This confirms the concurrent validity of the MALMAS. The sensitivity of the MALMAS was 88.9% while its specificity was 29.6%. The findings of this study further substantiates the reliability and validity of the MALMAS, in particular its concurrent validity and sensitivity for assessing medication adherence of people with type 2 diabetes in Malaysia. The World Health Organization (WHO) defined medication adherence as “the extent to which a person’s behavior in terms of taking medications, following diets or executing lifestyle changes, corresponds with agreed recommendations from the healthcare provider” . AlthougDepending on the complexity of the prescribed medication regimens, generally 40% of patients do not adhere to their medications . When coStudies found that adherence to oral anti-diabetes agents varied between 30% and 98%. This wide variation may be attributed to the different definitions used, duration of study, outcome measures and assessment methods, types of treatment and number of medications used –11. In dMedication non-adherence is also a costly economic burden worldwide ,12–14 wiPotential determinants of medication adherence could be divided into factors related to patient, medication and the healthcare system . These iAccording to the WHO, “Poor adherence to long-term therapies severely compromises the effectiveness of treatment making this a critical issue in population health both from the perspective of quality of life and of health economics” . For insA qualitative study conducted by Manias deduced that in general, patients perceived the clinician as a prescriber and one who manages their chronic conditions. The pharmacist, on the other hand is perceived as a central educator to assist patients in managing their chronic conditions and medication regimens as well as any issues related to adverse effects and medication adherence . NonetheAlthough many studies had been conducted to improve medication adherence, currently there is no gold standard available to assess this issue ,15. VariThe Morisky, Green and Levine Scale has been commonly used for the assessment of medication adherence ,31–33. TLu et al demonstrated that self-reported medication adherence was dependent on the recall period and a one-month duration was deemed practical . TherefoAlthough the MALMAS has undergone a validation process previously , severalThe MALMAS was developed based on the MMAS with 9 items (MMAS-9) . One of The first item of the MALMAS has five responses: (1) All the time, (2) Often, (3) Sometimes, (4) Rarely and (5) Never. The remaining seven items have a dichotomous response of “Yes” or “No”. MMAS-9 has not been validated and hence, the MALMAS was compared with the validated MMAS-8 to assess its convergent validity . The resA research team consisting of 12 experienced pharmacists developed and established the face and content validity of the MALMAS. In addition, a pilot study was conducted on five patients to obtain their feedback on the clarity of the MALMAS .People with type 2 diabetes, aged 21 years and above, who were using at least one anti-diabetes agent and could communicate in English, were recruited from the diabetes clinic of a major teaching hospital in Malaysia from April to May 2013. Based on convenience sampling, any person who met the inclusion criteria and visited the diabetes clinic during the study period would be approached to participate in the study while he/she was waiting to see the doctor. A researcher explained the purpose and study procedure to potential participants and requested them to complete a set of questionnaires, which consisted of the MALMAS and the MMAS-8. The participants took approximately 5 to 10 minutes to complete the questionnaires.The researcher counter-checked the completed questionnaire to ensure that all the questions were answered. The same participants answered the questionnaire again four weeks later. The Medical Ethics Committee of the University Malaya Medical Centre approved the study in writing (MEC Ref. No. 757.112). The study was conducted according to the principles expressed in the Declaration of Helsinki. All participants provided their written, informed consent upon recruitment into this study.2) test, while Wilcoxon signed ranks test or Mann-Whitney U test was used for testing the difference in continuous variables between two groups.All data were analyzed using the PASW Statistics for Windows, Version 18 . Using the Kolmogorov-Smirnov test, data obtained from both the MALMAS and the MMAS-8 did not fulfill the normal distribution criteria. Any possible associations between categorical variables were analyzed using chi-square , fair correlation (0.25–0.5), moderate to good correlation (0.5–0.75) and very good to excellent correlation (> 0.75) .1c values, using chi-square test, Kruskal-Wallis H test and Mann-Whitney U test. The correlation between the total score of the MALMAS and the HbA1c values of the participants was determined using Spearman’s rank order correlation coefficient.Concurrent validity was assessed by analyzing the levels of medication adherence and participants’ HbAThe sensitivity and specificity as well as the positive and negative predictive values of the MALMAS were generated. The sensitivity of the MALMAS determines its ability to correctly predict poor glycemic control in patients who are non-adherent to their medications, while the specificity of the MALMAS determines its ability to correctly predict good glycemic control in patients who are adherent to their medications . PositivA total of 136 participants were recruited in this study and their demographic and clinical data are presented in The MALMAS and MMAS-8 produced Cronbach’s alpha values of 0.565 and 0.595, respectively. However, the Cronbach’s alpha of MALMAS would increase to 0.571 if item 3 was excluded; while for MMAS-8, this value would increase to 0.598 if item 6 was deleted . The iteThe Spearman’s rho (ρ) for the test-retest results were 0.412 (p<0.001) for MALMAS and 0.376 (p<0.001) for MMAS-8. When the individual items in the MALMAS were analyzed, two out of the 8 items were significantly different at test retest while for the MMAS-8, three items were significantly different.The results obtained with the MALMAS and that with MMAS-8 showed no significant difference . A moder1c were significantly different between the levels of medication adherence, irrespective of how these levels were grouped together  was only observed when medication adherence was classified into two categories: high or medium adherence (considered as the adherence group) and low adherence (considered as the non-adherence group). The odds of a person with medium or high adherence achieving glycemic control is 3.36 (95% CI = 1.09–10.37) times more than those with low adherence .The median values of HbAtogether . Howeverdherence . In addiThe association between medication adherence and glycemic control is shown in The sensitivity and specificity of the MALMAS was 88.9% and 29.6%, respectively. The positive and negative predictive values were calculated as 31.7% and 87.9%, respectively.This study further established the psychometric properties of the MALMAS in terms of its acceptable internal consistency, stable reliability, concurrent validity and high sensitivity. This implies that the MALMAS is a reliable and valid instrument for assessing adherence to medications.The Cronbach’s alpha of the MALMAS was more than 0.5, which indicates that the MALMAS has acceptable internal consistency . The CroSix out of eight items in the MALMAS were not statistically different at test-retest. In addition, Spearman’s rho for the overall scores of the MALMAS showed fair correlation between the test-retest. This indicates that the MALMAS has achieved stable reliability.The prevalence of medication non-adherence determined using the MALMAS was similar to that of the MMAS-8, confirming the convergent validity of the MALMAS. In addition, these two instruments showed moderate to good correlation. These are similar to that of previous studies that assessed medication adherence using the MMAS-8 –45. Both1c values and the total MALMAS scores of the participants indicates that it is better to categorize the total MALMAS scores into two groups: adherence and non-adherence, rather than using the actual score to assess medication adherence. The odds of participants who were adherent to their anti-diabetes medications and achieving good glycemic control (HbA1c less than 7%) was 3.36 times of those who were non-adherence.The weak inverse correlation between HbA1c levels than those who were adherence. This confirms the concurrent validity of the MALMAS and the findings of previous studies where improving medication adherence would lead to better glycemic control in people with diabetes [1c by 0.16% [Participants who were non-adherence also had significantly higher median HbAdiabetes –47. Prevby 0.16% –47. TherThe MALMAS would be considered as an accurate instrument if a high number of true positives and true negatives were obtained, when compared to false positives and false negatives . The senThe high sensitivity and low specificity of the MALMAS is probably due to the small sample size ,49 or unOne of the limitations of the present study was that the MALMAS was validated only in English. Thus, only patients who could communicate in English were recruited. However, Malaysia is a multiracial society, where other languages such as Malay, Mandarin and Tamil are also widely used. Therefore, there is a need to translate and validate the MALMAS in these languages so that studies on medication adherence in Malaysia can include participants from different ethnic groups, which will better represent its multiethnic population.The concurrent validity of the MALMAS shows significant association between medication adherence and glycemic control while the other psychometric properties of the MALMAS conformed to that of other studies. In addition, the MALMAS has high sensitivity for identifying those who were non-adherent to their medications and had poor glycemic control. Therefore, this study further substantiates the reliability and validity, in particular the concurrent validity and sensitivity of the MALMAS for assessing adherence to long-term medications where a recall period of a month is required.