Health-related food taxes and subsidies may promote healthier diets and reduce mortality. Our aim was to estimate the effects of health-related food taxes and subsidies on deaths prevented or postponed (DPP) in New Zealand.A macrosimulation model based on household expenditure data, demand elasticities and population impact fractions for 18 diet-related diseases was used to estimate effects of five tax and subsidy regimens. We used price elasticity values for 24 major commonly consumed food groups in New Zealand, and food expenditure data from national Household Economic Surveys. Changes in mortality from cardiovascular disease, cancer, diabetes and other diet-related diseases were estimated.A 20% subsidy on fruit and vegetables would result in 560  DPP each year . A 20% tax on major dietary sources of saturated fat would result in 1,500  DPP (5.0%), and a 20% tax on major dietary sources of sodium would result in 2,000  DPP (6.8%). Combining taxes on saturated fat and sodium with a fruit and vegetable subsidy would result in 2,400  DPP . A tax on major dietary sources of greenhouse gas emissions would generate 1,200  DPP annually (4.0%). Effects were similar or greater for Maori and low-income households in relative terms.Health-related food taxes and subsidies could improve diets and reduce mortality from diet-related disease in New Zealand. Our study adds to the growing evidence base suggesting food pricing policies should improve population health and reduce inequalities, but there is still much work to be done to improve estimation of health impacts. Poor diets account for a substantial proportion of death and disability worldwide, with the major dietary risks being low fruit intake and high salt . The gloIn New Zealand, almost one third of adults 31%) and one in 10 children (11%) are obese 1% and on. ObesityHealth-related food taxes and subsidies, where the price of unhealthy foods is increased and/or that of healthy foods is decreased, are a potential means to promote healthier diets –9. In 20Systematic reviews suggest that these types of health-related food taxes and subsidies would be associated with beneficial dietary changes, and show potential for improved health –14. NatuOur objective was to estimate the effects of health-related food taxes and subsidies on mortality from diet-related diseases in New Zealand. We specifically aimed to incorporate any impact of compensatory food purchasing (via inclusion of both own- and cross-price PE in models); to use New Zealand-specific PE estimates because different PE values may lead to different modelling results between countries; to incorporate uncertainty around both PE estimates and epidemiological parameters in the model; and evaluate effects by income and ethnicity.Taxation and subsidy scenarios  were selWe modelled flat rate taxes of 20% applied to the retail price of major food group contributors to targeted nutrients , e.g. thSelection of food taxes and subsidies for modelling was constrained to some extent by the expenditure data used to define baseline food purchasing habits. Within the national household food expenditure datasets, nutritionally diverse foods are often aggregated into a single category, e.g. all milk and dairy products (both lower and higher fat options) were combined in one category. This meant the saturated fat tax model was applied to both reduced-fat and regular dairy products, rather than the more ideal scenario of full-fat dairy products only.The various data sources for the model are outlined in We used PE estimates to simulate changes in total food purchasing if selected foods were taxed or subsidised. PEs were estimated for 24 New Zealand food groups using data from HES 2007/08 and 2009/10 ,19 that To maximise model stability, each of the five taxation/subsidy models used own-PE and cross-PE values for the food groups targeted in each specific scenario and all theoretical substitutes and complements  instead We modelled changes in nutritional risk factors on mortality from diet-related diseases using the PRIME comparative risk assessment macrosimulation model . PRIME m95% uncertainty intervals (95% UI) are generated by Monte Carlo simulations (n = 5000) to reflect uncertainty around the input parameters of price elasticities and relative risks. The PRIME model has been used to model the effects of dietary changes on obesity rates and mortality due to chronic disease in the UK ,37–39, IA number of key assumptions underpinned our modelling analyses. These were: (1) changes in risk associated with changes in more than one food component were combined multiplicatively ; (2) with the exception of obesity, which follows a J-shaped curve, changes in risk followed a log-linear dose-response relationship; (3) relative risks were consistent across income groups and ethnic groups; (4) changes between baseline and counterfactual food and nutrient consumption distributions were made by all individuals within the population equally; (5) the tax/subsidy pass through rate to the consumer was 100%; and (6) counterfactual dietary intakes were rescaled to maintain the same total baseline dietary energy intakes where fruit and vegetable intakes increased (fruit and vegetable subsidy scenario only) to reflect evidence that increased fruit and vegetable consumption does not increase energy intakes .Population demographics were obtained from the 2006 New Zealand Census of Population and Dwellings . PopulatOwn-PE and cross-PE values derived for ethnic and income sub-groups proved to be unstable and highly variable so the impact of health-related food taxes and subsidies on consumption was estimated in two ways. The primary analysis applied the selected theoretical own- and cross-PEs for the total New Zealand population to all income and ethnic sub-groups. Differences in results for these subgroups is therefore due to differences in baseline diets and mortality between groups. A sensitivity analysis included the same theoretical own- and cross-PEs in the models but increased the own-PE values by an average of 30% for low-income households and 26% for Māori households to reflect known significant differences in New Zealand consumer demand by income and ethnicity .DPP in tables are presented as unrounded estimates, however in the text estimates are reported to the nearest two meaningful digits to reinforce the uncertainty inherent in the modelling.The baseline New Zealand population diet estimated from household food expenditure data was low in fruit (136g/day), vegetables (201g/day) and fibre (16.4g/day), and high in saturated fat (14.7% energy) and sodium  compared with national dietary targets to reduce chronic disease risk . There wOur model predicts that a 20% subsidy of price of fruit and vegetables would increase total population fruit and vegetable purchases by approximately 12% (95% UI 9.6% to 13.6%) and 18% (95% UI 16.5% to 18.9%) respectively, whilst saturated fat purchases would decrease by about 1% and sodium purchases would decrease by approximately 0.6% . Taxes oAll five scenarios modelled would avert or postpone deaths, with estimates ranging from 560 lives saved every year in New Zealand (95% UI 400 to 700) for a fruit and vegetable subsidy (assuming no change in energy consumption), to 2,400 lives saved  with a combination of taxes on foods high in saturated fat and sodium and a subsidy on fruit and vegetables . For allrelative terms except the fruit and vegetable subsidy, which would favour non-Māori , which is likely to have under-estimated income and ethnic sub-group effects given that lower-income and Māori consumers exhibit greater sensitivity to changes in the price of most foods . InternaWe modelled the potential effects of health-related food taxes and subsidies on mortality from diet-related non-communicable disease in New Zealand and found that taxes on unhealthy foods, subsidies on healthy foods, and their combination could prevent or postpone substantial numbers of deaths. Effects would be similar or greater for Māori and low-income households in relative terms, and likely greater for Māori and low-income households in absolute terms (DPP per capita within age groups).Our study has several strengths. We used the most recent New Zealand-specific food price elasticity and food expenditure data; we considered substitution to other foods; population disease-specific mortality rates were stratified by age, sex, income and ethnic group; and baseline diets were stratified by income and ethnicity. The macrosimulation model employed has been used previously to undertake similar analyses for the UK ,37–39, IHowever, there were some data limitations and necessary assumptions we had to navigate in order to address our objectives. Generation of accurate food PEs is challenging because it requires health policy-relevant groupings of food, accurate price and consumption data, and sufficient variation in price . The PEs we derived for our models are the best available national estimates but were obtained by linking the economic survey dataset to another dataset, the Food Price Index . They alPreviously we derived and published PEs by ethnic and socioeconomic group , but becFurthermore, to maximise model stability we made theoretical assumptions about which cross-price elasticity values to include in the models instead of using the full PE matrix for all 24 food groups. Our assumptions were based on the best available international evidence , but it The price elasticities we estimated were ‘conditional’, meaning that the estimation assumed that the total budget share for food did not change. This is a plausible assumption when a single food group undergoes a price change (e.g. a fruit subsidy), but becomes more tenuous when multiple foods undergo price changes, such as with a saturated fat tax. Future research could examine whether allowing for changes in food budget share  alters findings. Furthermore, reliable estimates of effects of such pricing policies on household expenditure should allow for change in budget share in line with non-trivial changes in the average price of foods.It is simple to express health gains in terms of deaths prevented or postponed, or lives saved. However quantification of the health gain in terms of health or quality adjusted life years gained would be better for two key reasons: it captures impact on both quantity and quality of life years gained (a DPP for an 85-year old is very different from one for a 45 year old); and it allows the inclusion of differing background morbidity and mortality rates by sex, age and social group .Additionally, we used the accepted population impact fraction method and calculated deaths postponed in the initial year of policy implementation; it would however take years  or even decades (e.g. effects of weight loss on cancer), for the full health gain to be realized. Therefore, the results of our study (DPP) should be interpreted as though the tax or subsidy had been in place for several years.Finally, there is uncertainty regarding potential supply-side responses to taxes or subsidies, including product reformulation to avoid taxes on specific nutrients, use of countervailing marketing campaigns or price promotion strategies  to limit (or amplify) the effects of such pricing policies, and variation in tax/subsidy pass-through rates to the consumer. Testing the sensitivity of results to such supply-side factors is challenging but we were able to do so for tax/subsidy pass-through rates. We assumed that the tax/subsidy pass-through rate to the consumer was 100%. In reality this may not be the case since food manufacturers or retailers might choose to absorb some of the increase in price or increase price beyond the minimum required. We did however assess the sensitivity of our results to plausible variation in the 'base case' tax/subsidy pass-through rate by modelling pass-through rates of +/-20% and, as expected, the effects of taxes/subsidy on mortality were modified accordingly i.e. increased or decreased by approximately 20%. For example, a pass-through rate of 0.8 in the saturated fat tax scenario would result in 1200 DPP each year, versus 1451 DPP with a pass-through rate of 1.0 and 1683 DPP with a pass-through rate of 1.2.There is global interest in health-related food taxes and subsidies to improve population diets and health ,27. CostAll five of the tax and subsidy scenarios we modelled could prevent or postpone deaths in New Zealand. However a similar UK study that modelled the potential health outcomes of four health-related food tax and subsidy regimens for the UK reported that two taxation scenarios  would have little effect, or even adverse effects on mortality rates in the case of the saturated fat tax . In contHealth-related food taxes and subsidies would reduce total population mortality from diet-related disease in New Zealand. Population groups likely to benefit most from such policies are Māori and low-income New Zealanders because their diets are currently less healthy than those of non-Māori and high-income New Zealanders, they experience a greater burden of diet-related disease reflecting their higher average BMI and higher risk of cardiovascular diseases and diabetes , and areWith any food pricing policy there is a risk of unintended consequences such as shifts from taxed foods to others that are equally or even more unhealthy. Our models suggest that a sodium tax could increase saturated fat purchases (by 3%). Such cross-PE effects could offset positive effects of health-related taxes but in this case the overall effects on population mortality rates remained positive. Consideration of the full implications of tax and subsidy packages is, therefore, critical.Relative to other strategies to prevent obesity and diet-related disease, health-related food taxes and subsidies are likely to be highly cost-effective. Previous studies found that taxes on unhealthy foods and beverages would be cost-saving and considerably more cost-effective than individually-focussed weight reduction programmes or community or school-based education programmes . AlthougHealth-related food taxes and subsidies could improve diets and reduce mortality from diet-related disease in New Zealand. However, there are many uncertainties in such modelling, not all of which are captured within the reported uncertainty intervals e.g. potential healthier product reformulation by industry in response to taxes and subsidies, which may enhance health gains. Our study adds to the growing evidence base that food taxes and subsidies should improve population health and reduce inequalities, but there is still much room for improvement in the estimation of health impacts.S1 Table(DOCX)Click here for additional data file.


In parallel, many computational methods have been developed to analyze and interpret these data. The interplay between RNA secondary structure and RBP binding has also been a growing theme. Integrating RNA–protein interaction data with observations of post-transcriptional regulation will enhance our understanding of the roles of these important proteins.RNA-binding proteins (RBPs) are important regulators of eukaryotic gene expression. Genomes typically encode dozens to hundreds of proteins containing RNA-binding domains, which collectively recognize diverse RNA sequences and structures. Recent advances in high-throughput methods for assaying the targets of RBPs  RNA-binding proteins (RBPs) have diverse roles in post-transcriptional gene expression, including regulation of alternative splicing, RNA export and localization, RNA stability and translation . Many RBin vitro and in vivo, as well as methods to determine the proteins bound to an RNA molecule. Finally, we discuss computational methods for analyzing high-throughput data and predicting RBP binding.Here we introduce the major classes of sequence- and structure-specific RNPs, and discuss aspects of RNA secondary structure that impact RBP binding; knowledge of how proteins interact with RNA is important for the interpretation of high-throughput data. We then review current methods for high-throughput experimental determination of the RNA targets of RBPs Different classes of RNA-binding domains (RBDs) use different strategies for binding to RNA. Nonetheless, there are some general features of RBP–RNA interactions: RNA recognition is commonly a combination of recognition of the RNA by the overall protein fold (involving hydrogen bonds with backbone atoms) as well as specific amino acid side chain–nucleotide interactions. Target specificity is often accomplished by way of hydrogen bonding and electrostatic interactions; the latter also contribute to the protein’s affinity for RNA along with stacking interactions . IndividThe RNA recognition motif (RRM) is found in all domains of life; in metazoans, it is the most common RNA-binding domain. A single RRM spans ∼90 amino acids and contains two conserved motifs, RNP-1 and RNP-2, consisting respectively of 8 and 6 mostly positively charged or aromatic amino acids , 11.Structurally, the RRM consists of a four-strand antiparallel β-sheet backed by two α-helices. RRMs are highly versatile in their mode of RNA recognition: in canonical RRM–RNA interactions, the bound RNA lies across the β-sheet and contacts one or more key residues in the conserved RNP-1 and RNP-2 motifs; however, the amount of the β-sheet surface directly contacting the RNA varies considerably . NoncanoThe β-sheet surface of a single RRM can contact up to four nucleotides, while engaging the loop regions external to the β-sheet can allow binding of up to six nucleotides . ApproxiK homology (KH) domains, named after the founding member of the family, hnRNP K , are alsDouble-stranded RNA binding domains (dsRBDs) are involved in diverse aspects of post-transcriptional regulation, including RNA editing , miRNA bBecause of the nature of the A-form RNA double helix, in which the major groove is narrow and deep, it is gIn contrast to the other major RNA binding domains, for which the elucidation of a recognition code has proven elusive, RNA recognition by Pumilio Homology Domains (PUM-HD) is understood to the extent that custom proteins can be designed to bind new sequences. The PUM-HD typically consists of 8 PUF (Pumilio and FBF) repeats of a 36 amino acid motif, and the entire domain forms a curved structure that binds RNA in the concave side of the domain, while the convex side mediates protein–protein interactions . Each PUZinc fingers are a large and diverse class of domains with the common property of coordinating zinc. The different types of zinc fingers have varying three-dimensional structure and likely have independent evolutionary origins. Nonetheless, several types act as DNA-, RNA-, and protein-binding domains. The extent to which individual zinc finger proteins bind and recognize each class of biopolymers is unknown, however there are some trends: C2H2 zinc fingers are usually DNA binding, while CCCH zinc fingers are primarily single-stranded RNA binding. CCHC zinc knuckles in viral and metazoan proteins also bind RNA; however, RNA binding by metazoan CCHC zinc knuckles is understood only in the context of proteins that also contain another RBD , 34. In The human genome encodes ∼60 proteins with CCCH zinc fingers (a larger number than contain KH domains), of which 11 have evidence of single-stranded RNA (ssRNA) binding . RNA recin vivo RNA structure is impacted both by the presence of ATP-dependent RNA helicases, as well as a number of proteins that could bind co-transcriptionally. Despite the development of high-throughput methods for measurement of RNA structure, there is ongoing controversy regarding the degree of RNA structure present in cells and the accuracy of both computational algorithms and these experimental techniques to predict RNA folding.RNA structure is a critical aspect of describing, measuring and predicting RBP binding: many RBPs recognize specific structures, while those that bind single-stranded RNA presumably compete with RNA structures. In addition, RBP binding likely has some impact on RNA structure, and in vivo in Arabidopsis [The secondary structure of an RNA molecule can be determined by footprinting techniques: cutting the RNA using RNases specific to ssRNA or dsRNA, or small molecule reagents that cleave or modify RNA at positions in a manner proportional to their accessibility . The clebidopsis  and yeasbidopsis .Arabidopsis using DMS [Arabidopsis using DMS [The overall trends identified by these studies vary. RNA accessibility around the start codon was associated with translational efficiency (as measured by ribosome profiling) in yeast using PARS  and in Asing DMS ; howeversing DMS . Using Psing DMS . This cosing DMS  as well sing DMS  all of wsing DMS  support Computational prediction of mRNA secondary structure generally conforms to one of the two approaches. The first relies on the assumption that thermodynamically stable structures are more likely to exist than unstable structures, exemplified by the Zuker MFOLD algorithm  and exteAs an alternative to the free-energy based algorithms, covariation-based approaches take advantage of the fact that functional RNA secondary structures are more likely to be conserved through evolution. Covariation algorithms use a number of simplifying heuristics (reviewed in ), as simGiven the inconsistencies among the experimental methods for assessing mRNA secondary structure and uncertainty about the accuracy of computational predictions, it is important to evaluate the accuracy of both these types of estimates. However, doing so has been troublesome because of the lack of gold standards for mRNA secondary structures. Classic RNA secondary structure benchmarks are likely inappropriate because they are composed of highly structured ncRNAs like ribosomal RNAs and ribozymes. In addition, mRNAs are longer than most well-characterized ncRNAs, such that windowed approaches  are often preferred both for their speed and potentially increased accuracy .Lange and colleagues performed an analysis to determine the accuracy of predicted secondary structures using yeast PARS data  and a cuin vitro approaches , also known as r an RBP , 63. In ratories . In addiratories . While Sratories , 63, andCaenorhabditis elegans PUM domain RBP FBF-2 to a 20 nt randomized pool alone and in the presence of a non–RNA-binding peptide fragment of the CPEB protein CPB-1, and found that FBF-2’s binding specificity was altered in the presence of the CPB-1 fragment. As well, they reported an alternate binding mode for FBF-2 with a slightly different motif.SEQRS is a method that applies high-throughput sequencing to SELEX to monitor the enrichment of optimal and alternate binding motifs by sequencing after each round of selection . SimilarRNAcompete  is a metNeither SEQRS nor RNAcompete, in their current versions, are optimal for determining the structural specificity of RBPs. SEQRS uses a 20 nt pool, which is not long enough to encompass all but the simplest of secondary structures, while RNAcompete, owing to its microarray-based strategy, is able to robustly represent only primary RNA sequence in the ∼244 k RNA sequences in the pool, and so the current version of the RNAcompete pool was designed to avoid highly structured sequences. Nonetheless, motifs determined for proteins known to prefer hairpin loops, such as yeast Vts1p and human SNRPA and LIN28A, were determined, and correspond to the single-stranded portions (e.g. the loop portion of a hairpin loop) of the structural motif. Significant preferences of many RBPs for ssRNA and, in a few cases, hairpin loops were identified . An alteTo study the kinetics of RBP binding to RNA sequences in a high-throughput manner, a recent study from the Greenleaf laboratory adapted an Illumina sequencing machine to measure binding affinity of the MS2 coat protein to ∼120 000 RNA sequences  represenin vivo by an RBP. The first genome-wide analyses were microarray based, and involved immunoprecipitation of RBP–RNA complexes using an antibody to the endogenous protein or to an epitope tag . Tenenbaum et al were the first to apply RIP-chip to the determination of the RNA fraction bound to HuB, HuA/HuR, eIF-4 E and PABP [A series of developments over the past decade have revolutionized determination of the RNAs bound and PABP . Since tand PABP  and reviand PABP ), leadinand PABP .in vitro after cell lysis, which has been observed under some conditions [in vivo contacts are maintained, as well as narrowing down the binding site [Although RIP allows identification of the target RNA molecules binding to an RBP, the data may include indirectly bound sequences, and precise locations of the binding site on the target mRNA may be difficult to determine. Additionally, RIP conditions must be calibrated to minimize reassociation of RBPs with mRNA nditions  but not nditions . Cross-ling site . UV crosing site , and so ing site  based oning site . The behing site . Preciseing site . In PAR-in vitro affinity measurements [C. elegans [C. elegans germ line [CLIP-seq and its variants are not without biases. UV cross-linking preferentially bonds certain nucleotides and certain amino acids , and noturements . UV crosurements , and who elegans  animals,erm line .in vitro are enriched in CLIP-seq reads [de novo [Recently, Friedersdorf and Keene demonstrated that a large fraction (up to 45%) of reads (including high-abundance sites) from published PAR-CLIP data sets overlap with binding regions observed in background data from FLAG-GFP immunoprecipitations . Backgroeq reads , often m[de novo . Backgro[de novo .Identification of protein-bound sites from CLIP-seq (and variants) data is nontrivial. Because transcript abundance varies, ascertainment of the protein’s preference for a target RNA through quantitation of the number of CLIP-seq reads mapping to a transcript sequence requires normalization to transcript abundance. Additionally, the choice of RNase and conditions chosen to fragment the cross-linked protein–RNA complex has significant impact on the base composition of the observed CLIP tags . To overin vivo methods discussed above to be protein-agnostic allows the identification of all the protein-bound sites in expressed transcripts.While the majority of recent efforts have focused on identification of the RNAs bound to a given RBP, mass spectrometry has allowed complementary approaches to discover new RBPs and determine all the RBPs binding an RNA. In addition, modification of the in vivo binding assay [in vitro or in vivo analyses are required to establish whether or not these novel RBPs simply bind RNA non-specifically as the PAR-CLIP data from many of the RBPs resembles background data derived from FLAG-GFP immunoprecipitations [Proteomics approaches have been applied to identify a more complete complement of RBPs in eukaryotic genomes, which is important because there are a number of RBPs without canonical RNA-binding domains , 92. In ng assay . Surprisitations .in vitro, and involve tethering the target RNA molecule to a solid support by chemical modification of the RNA [in vivo assembled RNPs has been accomplished using oligonucleotides complementary to the RNA sequence [Complementary to the RBP-centric approaches described above, development of mass spectrometric methods for determining the protein complement of diverse mixtures has allowed the direct ascertainment of proteins bound to an RNA sequence. Most approaches have been performed  the RNA  or using the RNA  and incusequence , coexpresequence , and delsequence . Althougsequence  and to csequence , demonstsequence .Several methods have been developed for discovering protein-bound sites on RNAs agnostic of the RBP, denoted protein occupancy (or interaction) profiling. These methods function similarly to PAR-CLIP, except that instead of immunoprecipitation of a target RBP, RNPs are purified using oligo (dT) beads  or chemiin vivo data will also be described.The goal of complete understanding of the functions of RBPs in post-transcriptional regulation requires computational analysis of RBP-RNA interactions to interpret experimental data and model how RBPs find and bind to their targets. A general strategy for predicting RBP binding involves (1) motif finding and (2) prediction of binding sites using the discovered motifs. Online databases collecting RBP motifs and Learning RBP binding motifs can be accomplished by applying DNA motif finders, which only consider the RNA sequence, or by considering RNA secondary structure either explicitly or as a layer on top of sequence preference. The basic approaches are summarized in Specialized methods that incorporate RNA secondary structure generally break down into two camps: the first is based on determining the linear structural context around a sequence motif. MEMERIS is an extension of the popular MEME algorithm that uses RNA accessibility as a prior probability to guide motif finding to single-stranded regions . SimilarThe second approach considers RNA secondary structure explicitly and includes stochastic context-free grammar (SCFG) and graphical approaches. CMfinder  and RNApThe fact that many RBPs have multiple RNA binding domains raises the interesting possibility of RBPs binding to bipartite or complex motifs. Gapped motifs have been described for PTB  and theIn vivo RBP binding sites are catalogued in three databases, starBase [Online databases that collect RNA–protein interactions are summarized in starBase , doRiNA starBase , and CLIstarBase . These dHigh-throughput identification of protein–RNA interactions has improved understanding of the targets of RBPs in diverse cellular contexts, and focus is shifting from cataloging RNA–protein interactions to understanding the individual and combined effects of RBPs on global RNA metabolism and gene expression. Methods to produce extensive functional data sets of RNA splicing , stabiliRBPs apply different strategies for binding RNA.For certain RBPs, RNA secondary structure is a key component of RNA binding, but widespread prediction and measurement of RNA secondary structure remains difficult especially due to the potential impact of other RBPs on mRNA structure.In vitro binding specificity of RBPs can be determined using microarray-based methods (such as RNAcompete) or high-throughput sequencing-based methods (such as RNA Bind-n-Seq).In vivo targets of RBPs are determined using CLIP-seq/HITS-clip, and precise binding sites are more easily defined using the PAR-CLIP and iCLIP variants.A general strategy for predicting RBP binding involves (1) motif finding and (2) prediction of binding sites using the discovered motifs.Canadian Institute for Health Research operating grant to Q.D.M. and T.R.H. MOP-125894.K.B.C. is supported by an NSERC Alexander Graham Bell Canada Graduate Scholarship. This work was funded by


The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed.Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform  Our laboratory, in a variety of experimental settings, has evaluated the Dox-regulatable Tet-ON system in rodents and NHP models using the rAAV platform. The general configuration of our vector construct consisted of a single expression cassette flanked by the AAV2 Inverted Terminal Repeat (ITR) sequences and containing the transgene of interest and the chimeric transactivator rtTA. The transgene of interest was placed under the control of the doxycycline-inducible Ptet-1 promoter, in which Tet operator (TetO) sequence concatemers are fused to the minimal promoter sequence derived from the cytomegalovirus (CMV) immediate-early promoter. In the same vector, the chimeric transactivator rtTA was under the control of either a constitutive ubiquitous or a tissue-specific promoter, such as the desmin promoter, when the latter construct was meant to be more specifically functional in the skeletal muscle E. coli (TetR) to the C-terminal portion of VP16 of herpes simplex virus (HSV). The rtTA epitope(s) - either originating from one of the two domains or both - recognized by the macaque immune system remains unknown, but if the transactivator domain of the molecule bears the dominant epitopes, then using a less antigenic transactivator protein would potentially be beneficial to support long-term transgene regulation.Long-term expression of transgenes can be supported by recombinant Adeno-Associated Virus (rAAV)-derived vectors in a variety of target organs in mammals, including human patients, after a single administration et al. generated a Dox-sensitive transrepressor called TetR-KRAB TetO sequences in the absence of Dox, thus suppressing the activity of the nearby promoter(s). In contrast, upon Dox administration, TetR-KRAB is sequestered from TetO sequences, allowing transgene expression after KRAB-mediated transcription repression is lifted in vitro gene expression switch even when low amounts of Dox were added to the culture . The TetR-KRAB drug inducible system has also been demonstrated to be an efficient, versatile tool in rodent models in the context of lentiviral vector-based gene transfer TetO-linked transcriptional units were repressed by TetR-KRAB only when integrated into the genome et al. in vitro and in vivo in a mouse model, using either integration-deficient lentiviral vectors or rAAV-based vectors. Recently, another study demonstrated the functionality of the TetR-KRAB repressor-based system after IM delivery of a rAAV2/8 vector in the mouse By fusing the KRAB domain of the human zinc-finger protein Kox1 to the DNA binding domain of the Tet repressor, Deuschle versus a macaque model. Because the human and putative macaque Kox1 nucleotide sequences are more than 95% homologous, we hypothesized that exchanging the HSV VP16 domain with the human KRAB one may result in less immunotoxicity than when the rtTA transactivator is expressed from the macaque skeletal muscle.In this study, we evaluated the ability of the TetR-KRAB system to mediate concise and reproducible transgene transcriptional regulation after subretinal rAAV delivery in rats. Because the efficiency and immunogenicity of the TetR-KRAB based-system has not been explored in higher species, we also evaluated the system after IM delivery in mice d2GFP reporter gene was under the control of the TetO-cytomegalovirus (CMV) entire natural promoter (CMVlg). The destabilized GFP variant d2GFP has a reduced half-life (2 hours instead of >24 hours for the standard GFP forward) or in the opposite (opposite) directions  compared with non-injected rats (data not shown).For retinal gene transfer, we used rAAV vectors in which the TetR-KRAB expression was driven by the ubiquitous CAG promoter and the via the drinking water, the GFP signal in the retina increased and reached a maximum within 14 days . The signal remained stable during the induction period of 3 weeks. Within 14 days after Dox withdrawal, the GFP signal decreased progressively to the baseline levels observed before induction . The kinetics of the regulation of transgene expression was similar for both groups of rats, either injected with the forward .Upon induction with 10 mg/kg/day of Dox by oral administration in vivo retinal imaging to monitor GFP expression kinetics ((data not shown). In contrast and similarly to the first induction, GFP expression increased upon Dox administration in the other rats within 14 days to a maximum, remained stable over 3 weeks (duration of induction) and decreased within 14 days upon Dox withdrawal. This persisting gene regulation was further confirmed by the quantification of GFP fluorescence, with a slighltly more stringent regulation achieved with the forward vector . Thus, TetR-KRAB responsiveness to doxycycline was maintained over time for up to one year for the forward and opposite vectors.All rats, except one control rat for each group, were subjected to repeated Dox exposures over a one-year period, and retinas were analyzed by kinetics Figure 2mEpo) gene driven by three different regulated ubiquitous promoters , and Epo reporter gene regulation was monitored by hematocrit values and circulating Epo levels measured by ELISA. A control group was injected with a rAAV1 vector in which the Epo reporter gene was under the control of the rtTA transactivator-dependent TetON system. Similar to the KRAB constructs, the two expressing units were also in the forward orientation, and the expression of the rtTA transactivator was driven by the CAG promoter. In the absence of Dox, the PGK TetR-KRAB group had the most repressed Epo expression; the mean of the Epo values was not higher than the basal Epo level in non-injected mice  ((Data not shown). In comparison to non-injected mice, a higher basal Epo expression in the absence of Dox was observed in all other groups 10 days after vector delivery. The CMVlg promoter resulted in the highest increase of hematocrit levels (Data not shown), according to the highest Epo background level, in the absence of Dox . Thus, the combination of the forward orientation and the TetO-PGK promoter allowed successful regulation with little to no background expression in the absence of Dox. The better regulation with the PGK promoter was not due to a difference in transduction efficiency as suggested by the number of vector copies quantified in the injected muscle . Indeed, while all the injected groups were statistically different from the mock non-injected group , no statistical difference was found between the PGK group and the other vectors . We further monitored the hematocrit and Epo levels in the PGK TetR-KRAB group at regular time intervals and after several Dox cycles over a period of 42 weeks post-injection  gene under the control of the TetR-KRAB protein. The optimal construction defined in the murine studies in terms of Epo transgene expression and regulation was used: the two-expression cassettes TetO.PGK- cmEpo-pA and CAG-TetR-KRAB-pA were cloned in the same (forward) direction . Furthermore, Mac 2 and Mac 3 also had a loss of inducible Epo expression following the second Dox cycle . As previously observed with the original rtTA-based regulatable system, the loss of gene regulation was associated with an immune response against the TetR-KRAB protein. The recognition of TetR-KRAB protein by the host immune system appears to be initiated earlier than we observed in our previous cohorts expressing the original rtTA transactivator Figure S3A , a finding that was correlated with the complete abolition of gene regulation in this animal. The humoral response against the TetR-KRAB protein was associated with muscular cell infiltrates at the sites of vector injections in all three macaques. These infiltrates were localized either in perivascular areas or surrounding some necrotic fibers and were predominantly made of mononuclear cells  and the rapamycin regulation systems and the mammalian steroid receptor (mifepristone and tamoxifen)- and the insect steroid receptor (ecdysteroid)-based systems. Tetracycline-dependent systems have been the most widely used in rodents with sustained transgene regulation. However, when translational studies were initiated in larger animals, the development of an immune response against the rtTA transactivator was observed, leading to transient gene regulation in vivo a recent Tet-dependent system based on the TetR-KRAB regulatory protein to determine if it exhibits the features required for clinical translation, including successful assessment in large animal models. With respect to potential rAAV-based gene therapy applications targeting the skeletal muscle or the retina, we first assessed the system in rodents after either rAAV5 subretinal administration or rAAV1 IM delivery. In both gene transfer models, the major features desirable for a gene regulation system were observed: (i) undetectable to low reporter gene expression in the absence of Dox and (ii) induction of expression in the presence of Dox that is fully reversible after Dox withdrawal. Similarly, previous studies have demonstrated efficient TetR-KRAB-mediated gene regulation in rodents using either lentiviral The clinical efficacy and safety of rAAV-based gene transfer will be improved with the use of systems able to turn “on” and “off” therapeutic gene expression. This is crucial for maintaining appropriate levels of gene products within the therapeutic range in order to prevent potential toxicity. It is also critical, for the ability to turn off gene expression in the case of harmful side effects. Four drug-dependent regulation systems have been described to date TetO sequences (forward orientation) or at a distance of 4 kB in the opposite construction. In our gene transfer model, the forward orientation was as active as its counterpart, with even a slightly better gene regulation than with the opposite construction, as demonstrated by d2GFP quantification. This indicates that the CAG promoter was not repressed by the KRAB box in our retinal model. We further confirmed this observation in the mouse muscle, where the forward orientation was also able to drive Epo gene regulation.Because the KRAB box has demonstrated the ability to inhibit all DNA Polymerases I, II or III within a range of 3 kB of its DNA attachment site Epo gene expression to evaluate whether concise inducible gene expression is dependent on the strength of the promoter used. The long and short CMV promoter versions resulted in higher Epo basal expression in the absence of Dox, in contrast to the PGK promoter, which resulted in similar Epo levels compared with control mice. The long CMV version, with its complete enhancer region, resulted in an eight-fold Epo leakage compared with basal Epo in control mice, versus only 2-fold for the short CMV promoter variant. Therefore, to obtain the most concise regulation and physiological expression upon induction, promoter choice appears critical. It is desirable to develop additional strategies to restrict gene expression in the target tissue using either a specific-tissue promoter or miRNA-regulated vectors. For instance, Pichard et al. have recently developed a strategy combining the TetR-KRAB artificial transgene-repressor, the endogenous miRNA silencing machinery and tissue specific promoters with optimized spatial and temporal gene expression control after rAAV8-based gene transfer in mice In the mouse muscle, we compared three different promoters driving (i) local change in chromatin structure, (ii) local histone deacetylation, and (iii) indirect influence of the arrangement of the basal transcription machinery. Therefore, until recently, the hypothesis was that only integrated promoters could be inhibited by TetR-KRAB The mechanism by which KRAB inhibits expression of genes is not entirely known. Three pathways seem to be involved: versus immunogenicity of the TetR-KRAB based-system has not yet been explored in higher species, we also evaluated the system after IM delivery in mice versus the macaque model. Immune reactions against the chimeric transactivators emerged as a main limitation upon translation of the tetracycline- and the rapamycin-regulation systems to large animals 11 vg per injection site and the number of in situ vector copies were comparable between Mac 1 and the rest of the cohort . Despite their more rapid production in Mac 1, IgG anti-TetR antibodies were only detectable following the first Dox induction (performed at 2 months pi). This finding suggests that TetR-KRAB protein recognition by the host immune system could first require its Dox-mediated conformational change.As the long-term efficiency Figure S4). Regarding our macaque cohort who received the TetR-KRAB construct, we were not able to demonstrate a T cell effector response against TetR-derived peptides in an IFNγ-ELISPOT assay after PBMC and splenocyte stimulation . This may be due to a transitory response, as T cells were harvested at the euthanasia of animals 2 years post-injection, or to the limited sensitivity of the assay. However, similar to our previous studies using the TetON system in the macaque Figure S2B). Additionally, we cannot exclude that the observed infiltrations were due to another component in the vector preparation or even the viral capsid itself.Exchanging the HSV VP16 domain of the rtTA transactivator with the human KRAB, which is over 95% homologous to the macaque Kox1 sequence, did not result in less immunotoxicity than when the rtTA transactivator was expressed from the macaque skeletal muscle. The host immune system may be recognizing the common TetR component that is of bacterial origin. Our data confirm this hypothesis, as we used the recombinant rtTA protein  for IgG antibody detection. Nevertheless, we are not able to conclude whether the KRAB component was also recognized by the macaque immune system. In our previous studies where the rtTA-protein was expressed in the macaque, we also obtained data suggesting that cellular immunity is directed against the TetR component of rtTA, not the viral VP16 protein. In an IFNγ ELISpot assay using an rtTA-derived peptide library, epitopes inducing IFNγ secretion were predominantly located within the first 110 amino-acids of rtTA, corresponding to the N-terminal of the TetR component. This was observed in 4 animals that received either a rAAV or an adenoviral vector expressing the rtTA transactivator  where the study was realized. This animal facility received the authorization #0056397-B delivered by the Departmental Direction of Veterinary Services . All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals and in accordance with the French law concerning experimentations on vertebrate laboratory animals . C57Bl/6 6 week old male mice and adult Wistar rats were obtained from Charles River laboratory (France), and captive-bred macaques (Macaca Fascicularis) were purchased from BioPrim . NHP experiments were conducted on 3–5 kg male macaques with no detectable neutralizing antibodies against AAV serotypes before inclusion in the protocol. Rats experiments were conducted under the agreement #B44–32 and mouse and NHP studies under the agreement #F44–273 delivered by the Departmental Direction of Veterinary Services . Macaque study was conducted in accordance with the recommendations of the Weatherall report: “The use of nonhuman primates in research”. The macaques were housed in an enriched environment (toys) and were under a varied feeding regimen that included fresh fruits and vegetables. In addition, they were monitored daily for health and welfare. To avoid any discomfort during and after the experiments, all procedures were carried out after animal sedation with 20 mg/kg of Medetomidine  and 8 mg/kg of ketamine . Intramuscular injections of rAAV vectors and intravenous injections of Doxycycline were classified as mild severity procedures. Vector administration was performed under ketamine/medetomidine-induced anesthesia maintained with 1,5% isoflurane. Primates were monitored by clinical observations, respiratory rate and temperature measurements, electrocardiography, oxymetry, and capnography. Analgesia was performed with 0,1 mg/kg morphine . During the protocol, special attention was paid to the health and welfare of animals, and blood samples were collected regularly to follow biochemical and hematological parameters, in addition to the clinical follow-up. At the end of the protocol, euthanasia was performed with intravenous injection of Sodium Pentobarbital  after 0,1 mg/kg morphine-induced analgesia.d2GFP or Epo reporter genes were under the control of either TetO-CMV (either short or long versions) or TetO-PGK promoters. Gene accession numbers for mEpo, cmEpo, KRAB and TetR sequences are BC119271.1, M 18089, NM015394 and BAG71042.1, respectively. The two expression units were cloned either in the same (forward) or in the opposite directions between the Inverted Terminal Repeats (ITRs) of AAV2. The viral recombinant vector used in the NHP study was approved by the Scientific Committee of the "Haut Conseil des Biotechnologies" of the French Ministry of Research under reference #4597CA-II.rAAV1 and rAAV5 viral preparations were all manufactured using the 293 cell transfection method and purified by cesium chloride density gradients followed by extensive dialysis against phosphate-buffered saline (PBS). The viral constructs are described in via a transscleral transchoroidal approach as previously described Materials and Methods S1.Wistar rats were anesthetized with an IM injection of ketamine and xylazine . Subretinal injections were performed 9 total viral genomes (vg) of vector was injected in the muscle in a volume of 30 µL. Dox  was dissolved in the drinking water to a final concentration of 200 µg/mL with 5% sucrose. Blood samples were obtained by retro-orbital puncture in isoflurane-anesthetized animals. Hematocrit levels were monitored weekly and serum Epo levels were measured before and after Dox inductions by enzyme-linked immunosorbent assay (ELISA) using Mouse Epo Quantikine ELISA kit . The quantification of vector copy numbers by qPCR in the muscles of mice was performed as described in Materials and Methods S1.IM injections in mice were given in the tibialis anterior muscle after anesthesia with ketamine and xylazine. In all groups, a dose of 3×1010 vg/kg split over 5 to 8 pre-tattooed injection sites along the two tibialis anterior muscles was administered in a volume per site of 500 to 650 µL. The induction protocol started 2 months after rAAV1-TetR-KRAB/Epo delivery. Doxycyclin  was given intravenously . Circulating Epo levels were measured by ELISA using the Human Epo Quantikine IVD kit . The quantification of vector copy numbers by qPCR in the muscles of NHP was performed as described in Materials and Methods S1.IM injection of rAAV in NHP was conducted as described previously Materials and Methods S1.Detection of anti-TetR antibodies was conducted as previously described Tibialis muscles were sampled at animal euthanasia 2 years post-injection. Hematoxylin eosin saffron staining (HES) was performed as per standard histological protocols using formol-fixed and paraffin-embedded muscle sections.Figure S1d2GFP fluorescence quantification in the retina after rAAV.TetR-KRAB/d2GFP administration.(PDF)Click here for additional data file.Figure S2Quantification of vector copy numbers in the muscle after rAAV.TetR-KRAB/mEpo intramuscular (IM) administration of rAAV vectors.(PDF)Click here for additional data file.Figure S3Monitoring of anti-TetR humoral and cellular immune responses in Mac 1, 2 and 3 after rAAV1-mediated TetR-KRAB expression.(PDF)Click here for additional data file.Figure S4Anti-rtTA cellular immune responses in the macaque are directed against epitopes among the TetR component and not the VP16 one.(PDF)Click here for additional data file.Materials and Methods S1Quantification of d2GFP fluorescence in the retina. Quantification of vector copy numbers by qPCR in the muscle. Follow-up of anti-TetR cellular immune responses in NHP.(PDF)Click here for additional data file.Checklist S1ARRIVES Checklist.(PDF)Click here for additional data file.


The beam of energetic ions randomized the atomic arrangement locally, leading to the formation of ferromagnetism in the ion-affected regime. The interaction of a penetrating ion with host atoms is known to be spatially inhomogeneous, raising questions on the magnetic homogeneity of nanostructures caused by ion-induced collision cascades. Direct holographic observations of the flux-lines emergent from the disorder-induced magnetic nanostructures were made in order to measure the depth- and lateral- magnetization variation at ferromagnetic/non-ferromagnetic interfaces. Our results suggest that high-resolution nanomagnets of practically any desired 2-dimensional geometry can be directly written onto selected alloy thin films using a nano-focussed ion-beam stylus, thus enabling the rapid prototyping and testing of novel magnetization configurations for their magneto-coupling and spin-wave properties.Nanomagnets form the building blocks for a variety of spin-transport, spin-wave and data storage devices. In this work we generated nanoscale magnets by exploiting the phenomenon of disorder-induced ferromagnetism; disorder was induced locally on a chemically ordered, initially non-ferromagnetic, Fe Magnetic nanostructures are of critical importance for spin-transport devices24Achieving the varied geometries and small sizes of magnetic objects, necessary for these applications, is challenging. For instance, spin-transport applications require magnetic nanostructures separated by non-magnetic conducting barriers. Such structures are typically hetero-structures containing several interfaces that result in spin-scattering losses. Moreover, these devices can require a number of patterning and electrical contacting steps. Similarly, magnonic devices may require wave-guides of various shapes and defined magnetization configurations to enable spin-wave propagationMs, of certain alloys can be drastically increased due to the chemical disordering8A solution to patterning magnetic nanostructures of desired geometries is to write magnetic objects directly onto a non-magnetic precursor by the use of an energetic ion beam. It has been shown that the saturation magnetization, Ms itself remains fixed or is reduced due to ion-induced damage of the crystal structure or alloying with the ion-species. The converse is true in certain cases, whereby magnetization is induced in regions that interact with the ions. A variety of mechanisms can be exploited such as ion-induced magnetization due to a fcc-bcc phase transitions in ultrathin Fe films+Ion-irradiation can modify a variety of magnetic properties in alloys, including the magnetization easy axes, magnetic anisotropy, coercive fields and ferromagnetic resonances1060Al40 thin films as our model system. This is a powerful way to directly write magnetic nanostructures using ions, as it is a physical process that involves the displacement of only a few atoms within the lattice to generate large magnetization, while preserving the flat topography of the original film. Alloys possessing disorder-induced ferromagnetism phenomena usually consist of atoms of magnetic and non-magnetic species, and magnetic phase transitions can be triggered by chemical disordering. That is, a chemically ordered thin film acts as a template onto which disordered regions can be printed using localized exposure to an ion beam. Ions penetrating through a chemically ordered structure form collision cascades, where disorder is formed locally, and ferromagnetism is generated due to an increase in the number of magnetic moment carrying nearest-neighbours141516Here we focus on ion-induced magnetization due to chemical disordering, taking FeTo produce magnetic nanostructures it is necessary to confine the volume over which the ions interact with the host atoms. One approach is to irradiate through lithographically patterned shadow masks81213+ or Ne+. For high resolution work the beam tails are reduced to a minimum by using a small aperture and placing the beam crossover at a large distance from the aperture. This ion-optics design ensures that ions originating from a single ionization site only  can reach the sample, resulting in a narrow beam of + ions. It is difficult to estimate the profile for such narrow beam widths60Al40 (State-of-the-art instruments such as the He-ion Microscope (HIM) can focus ions down to ed60Al40 .via direct observations.The distribution of atomic displacements caused by impinging ions tends to be spatially inhomogeneous. An ion penetrating a crystalline material undergoes deceleration due to the crystal potential and collisions with the nuclei, until the ion is stopped completely at a certain depth. Nuclear collisions can cause atoms to be knocked out from their lattice sites, which results in recoiling ions and atoms in arbitrary directions. The collision processes are inhomogeneous over the depth- and lateral- directions, and modifications to magnetic properties resulting from the atomic displacements, such as the strongly increased magnetization, may consequently also follow an inhomogeneous spatial distribution. Spatial inhomogeneity limits the resolution of the patterned magnetic objects and determines their functionality as device elements. The spatially resolved effect of low dose damage cascades on functional properties is typically challenging to visualize and with few exceptions60Al40. In case of continuous thin films which have their magnetization, M, confined to the film plane, the magnetic flux density within the film is given by μ0M. Therefore μ0M can be measured across the film thickness for the ion-magnetized films discussed here. The value of μ0M was observed to strongly vary depending on the ion-penetration depth.Observations of the depth- and lateral- distribution of the flux lines of nanostructured objects were performed using Transmission Electron Microscopy (TEM) combined with electron holography. Off-axis electron holography was employed to observe and quantify the depth- and lateral- distribution of the magnetic flux density of ion-magnetized structures in FeLateral magnetic patterning introduces boundaries within the sample where the continuity of the magnetization is broken and stray flux lines leak from the edges of the magnetic structures. Electron holography was employed to laterally trace the stray field in order to draw conclusions on the magnetization variation around the magnetic edge regions. It is shown that the stray flux density in the vicinity of edges of magnetic structures depends sensitively on the nanoscale sharpness of the edge.μ0M induced by ions of different energies penetrating Fe60Al40 thin films. Furthermore, the lateral-distribution of μ0M for bar magnet-like objects separated by 100 nm wide spacings is estimated. The direct confirmation of well-defined magnetic structures patterned using an ion-beam shows that ion-induced ferromagnetism is a powerful approach to pattern homogeneous and defined nanomagnets for a vast variety of applications.Electron holography revealed the depth-distribution of 60Al40 films that were uniformly irradiated with Ne+ ions at various ion-energies. In addition we also observed the magnetic flux-density within an unirradiated B2 ordered film as control sample. These measurements were necessary to show that the depth-profile of the induced magnetization is a function of the ion-energy, and above a certain energy the films can indeed be homogeneously magnetized using energetic ions, which can be important for patterning functional magnetic objects.Before studying magnetically patterned samples, we observed the depth variation of the magnetic flux distribution in 40 nm thick, B2 ordered Fe60Al40 films that were initially in the B2 phase, and subsequently irradiated with 5, 10 and 20 keV Ne+ ions with a fluence of 6 × 1014 ions cm−2; as well as a 100 nm thick Fe60Al40 film irradiated with 30 keV Ne+ ions with the same fluence.The depth distribution of the ferromagnetic A2 phase formed due to atomic displacements depends on the ion-energy; the depth of the magnetized region (or the A2/B2 phase boundary) with respect to the film surface can be expected to increase with increasing ion-energy. + fluence of 6 × 1014 ions cm−2 the maximum Ms is reached within the ion-affected regions, for all of the above ion-energiesM integrated over the whole sample volume, whereas here we perform local observations of the variation of The fluence is rather low such that ion-induced variations of the grain structure and morphology are negligiblemag. To help isolate the contribution of φmag it is necessary to apply magnetic fields insitu, which has been done using the magnetic field of the objective lensmag is usually multiplied by an amplification factor. Plotting cos(amplified φmag) directly visualizes the projected magnetic flux density within the sample plane. For each sample shown in +-ions at 5 keV . A 10 μm beam limiting aperture and a very short dwell time of 0.1 μs have been used to write the pattern at a current of 0.8 pA. To achieve the desired fluence while reducing inhomogeneities related to the low beam current each pattern was scanned several times. The resolution of the patterned structures is limited by the lateral scattering of ions.Next, we investigated a magnetically structured sample in an attempt to observe the lateral distribution of the flux lines. A separate sample was irradiated using a HIM with a nano-focussed Neμm wide wire using the nano-focussed beam. The ion-beam consisted of Ne+ ions at 25 keV i.e., with energy sufficient to fully penetrate the 40 nm thick Fe60Al40 film. The 2-dimensional stripe pattern consisted of 500 nm wide irradiated regions, separated by 100 nm wide spacings. Magnetic contrast was observed using the Kerr effect in an optical microscope , is shown in The sample region investigated was expected to cover three ferromagnetic stripes, with one in the centre and two half-stripes on the left and right side respectively. The ferromagnetic stripes were to be separated by 100 nm wide non-ferromagnetic spacers. The solid black line in The flux-line distribution of the non-ferromagnetic spacer region between the two well-formed stripes is shown in higher magnification in From the magnetic phase image of viz. in the non-ferromagnetic regions above and the substrate region below.The region corresponding to the half-stripe on the right remained non-ferromagnetic, as observed by the significantly reduced density and the incoherence of the flux lines in this region. Furthermore stray flux-lines do not originate from this region, as is the case with the two ferromagnetic stripes. The reason for the nearly vanishing flux-density of this region is unclear; however it is likely that the ion-beam missed irradiating this particular region.At the ferromagnetic-edge region shown in We return to the continuous thin film samples depicted in + irradiation at 5, 10 and 30 keV is shown in μ0M values shown on the vertical axis have been extracted from the gradient of the magnetic phase shift (see Methods), normalized by subtracting a background and dividing by the effective thickness of the sample in the measured region  along a line that runs horizontally along the film interface, in the vicinity of the ferromagnetic edge (solid red line in mag) distribution along the interface for To illustrate the sensitivity of the positions of the exit points on the magnetization profile, The clear differences in the positions of the exit points of the flux lines, for the two cases of The observations of this study are far more direct than the previous modeling work, and they help in achieving minimum possible non-ferromagnetic spacer distances. Narrow non-ferromagnetic spacers are of vital importance for spin-transport devices, wherein the spacing between two active ferromagnetic elements must be narrower than the spin-diffusion length. The width of the non-ferromagnetic spacing is also crucial in controlling the magneto-static coupling between neighbouring ferromagnetic elements at the nanoscale. Magnetically modulated materials consisting of arrays of ferromagnetic elements surrounded by narrow non-ferromagnetic spacers can show intriguing spin-wave resonances when pumped with microwave excitations. Such spin-wave or magnonic phenomena have been suggested as schemes for highly tuneable magneto-logic devices. Ion-induced ferromagnetism provides a convenient pathway to produce ferromagnetic elements with tailored magnetic coupling at the nanoscale for a variety of possible applications.μ0M where the magnetization, M, is induced by ions penetrating a system with disorder induced ferromagnetism. The depth distribution of μ0M can be controlled by varying the energy of the impinging ions. Homogenously magnetized thin films were formed by providing ions with sufficient energy to fully penetrate the film. Ferromagnetic nanostructures have been patterned using a nano-focussed ion-beam and the emergent flux line distribution observed using electron holography was used to fingerprint the magnetization profile in the vicinity of a ferromagnetic edge. The behavior of flux lines at the nanoscale can be modelled using Maxwell’s equations. This can open fascinating prospects in the field of nanomagnetism, whereby it may be possible to write nanomagnets of desired shapes onto materials that exhibit ion-induced ferromagnetism. Such structures may prove useful, for instance, as channels for spin-wave propagation and manipulation, as well as for lateral spin transport architectures. The flexibility with which the magnetic nanostructures can be written makes ion-induced ferromagnetism an ideal pathway for rapidly producing prototypes of novel magnetization configurations for use in future magnetic devices.Direct holographic observations have been made to map the depth- and lateral- distribution of 60Al40 thin films were deposited on a 150 nm thick SiO2 layer grown onto a silicon substrate. These films were prepared by magnetron sputtering and annealed for 1 hr at 773 K in vacuum. Annealing was necessary for the formation of the chemically ordered B2 phase, the existence of which was confirmed using X-ray diffraction, as well as Transmission Electron Microscopy. Unless otherwise stated, the film thickness was kept fixed at 40 nm. Disorder was created by concerted Ne+ ion irradiation at different kinetic energies ranging from 5–30 keVThe Fe+-ions nm–2 and to ensure a uniform distribution of the damage events.An Orion NanoFab Helium Ion Microscope (HIM) from Carl Zeiss AG has been used to pattern the nano magnets into the FeAl film. Helium Ion Microscopy utilizes a 0.5 nm wide beam of 30 keV Helium ions to visualize or structure samples at the nanoscale. Here, Neon has been used in the Gas field ion source+ broad-beam irradiated Fe60Al40 films glued together in face-to-face geometry using G2 epoxy glue (Gatan), were prepared by sawing , grinding , polishing , dimpling  and final Ar+ ion milling . To guarantee the reference wave passing through field-free space in the holography experiment, the classically prepared specimens were divided into two parts along the glue line with only one part being actually used for analysis.Classical TEM cross-sections of Ne+ion beam structured Fe60Al40 film was done by in situ lift-out using a Zeiss Crossbeam NVision 40 system. To protect the sensitive surface of the magnetically patterned film at the area of interest, a carbon cap layer was deposited beginning with electron beam assisted precursor decomposition and subsequently followed by Ga focused ion beam (FIB) assisted precursor decomposition. Afterwards, the TEM lamella was prepared using a 30 keV Ga FIB with adapted currents. The lamella was transferred to a 3 post copper lift-out grid (Omniprobe) using a Kleindiek micromanipulator. To minimize sidewall damage, Ga+ ions with energy limited to 5 keV were used for the final thinning of the TEM lamella to electron transparency.TEM lamella preparation of the NeWe apply Off-Axis Electron Holography25262829In the Wentzel-Kramers-Brillouin (WKB) approximation, the phase shift is given by:E is the interaction constant (7.29 V−1μm−1 for 200 keV electrons). To separate electric and magnetic phase shifts the method of in-situ magnetization reversal is appliedThe first integral denotes the projection of the electric potential V and the second of the magnetic vector potential el) image is given by the half sum and the magnetic phase (ϕmag) image is given by the half difference of the two phase images, respectively. The projected in-plane component of the magnetic induction is determined by the gradient of the magnetic phase image and dividing the thickness determined from the electric phase shift. The latter step assumes a homogeneous magnetic induction along the beam axis within the sample. The mean in-plane components are given by:The wave series were aligned to cancel object and biprism drift as well as biprism charging by means of the Triebenberg software packageHow to cite this article: Röder, F. et al. Direct Depth- and Lateral- Imaging of Nanoscale Magnets Generated by Ion Impact. Sci. Rep.5, 16786; doi: 10.1038/srep16786 (2015).


Manual segmentation is time consuming and operator dependent. Automated segmentation as usually performed via single atlas registration fails to account for anatomo-physiological variability. We present, evaluate, and make available a multi-atlas approach for automatically segmenting rat brain MRI and extracting PET activies.Preclinical 18F]FDG PET. Automated and manual segmentations were compared for morphometric performance (assessed by comparing volume bias and Dice overlap index) and functional performance .High-resolution 7T 2DT2 MR images of 12 Sprague-Dawley rat brains were manually segmented into 27-VOI label volumes using detailed protocols. Automated methods were developed with 7/12 atlas datasets, i.e. the MRIs and their associated label volumes. MRIs were registered to a common space, where an MRI template and a maximum probability atlas were created. Three automated methods were tested: 1/registering individual MRIs to the template, and using a single atlas (SA), 2/using the maximum probability atlas (MP), and 3/registering the MRIs from the multi-atlas dataset to an individual MRI, propagating the label volumes and fusing them in individual MRI space . Evaluation was performed on the five remaining rats which additionally underwent [Only the SA method showed volume bias. Dice indices were significantly different between methods (PF>MP>SA). PET regional measures were more accurate with multi-atlas methods than with SA method.Multi-atlas methods outperform SA for automated anatomical brain segmentation and PET measure’s extraction. They perform comparably to manual segmentation for FDG-PET quantification. Multi-atlas methods are suitable for rapid reproducible VOI analyses. Preclinical neuroimaging studies are increasingly performed on rodents, including rats. Positron emission tomography (PET) imaging in rat animal models, in particular, provides a tool for studying the pathogenesis and progression of neurological disease and for validating new radiotracers and therapeutic agents Analyzing functional images requires a corresponding anatomical image so as to identify brain structures. Anatomical correspondence is also required to quantify and model interactions between the PET ligand and its pharmacological receptor. Without automated anatomical identification, analyzing PET data relies on time-consuming and observer-dependent manual volume-of-interest (VOI) delineation. Automated delineation usually consists in normalizing images in a reference space, and segmenting the structures in this space using a standardized digital brain atlas. This requires a normalization process, a template  and a digital atlas.MRI templates of rat brain spatially aligned to the Paxinos and Watson anatomical atlas The present study introduces two methods for segmenting rat brain MRIs using multi-atlas dataset. This work includes the creation of an MRI template and the multi-atlas dataset. The accuracy of automated segmentations using a multi-atlas approach was tested against single atlas method in a morphological evaluation, and in a functional evaluation using combined MRI and PET data from a test group.18F]FDG PET and were used as the Test group in the evaluation procedure. This study was carried out in strict accordance with the recommendations in the guidelines established by the European Communities Council Directive of November 24, 1986 (86/609/EEC). The protocol and the full study were approved by the Committee on the Ethics of Animal Experiments of the University of Lyon (Permit Number: C2EA-42). All imaging sessions were performed under isoflurane anesthesia, and all efforts were made to minimize suffering.Twelve healthy Sprague-Dawley rats were included and underwent MRI. Seven rats (weight 250–350 g) constituted the Atlas group. Five rats (weight 281–330 g) additionally underwent dynamic FDG radiotracer . Images were reconstructed with attenuation and scatter correction by a 3D-filtered back-projection algorithm  and a zoom factor of 2, resulting in a reconstructed volume of 159 128×128-voxel slices, in a 49.7×49.7×126 mm bounding box, with 0.388×0.388×0.796 mm voxel size. Ten-minute static images from 40 to 50 minutes (PET-FDG) were reconstructed.The 5 anaesthetized Test group animals were placed in the PET scanner in a prone position immediately after MRI acquisition, using the same support. PET scans were acquired using a Siemens INVEON PET/CT scanner, with nominal in-plane resolution of ∼1.4 mm full-width-at-half-maximum in the center of the FOV Automated registration used the minctracc program The MRI template was constructed from images of the 7 Atlas group rats. The template creation procedure is illustrated in Display software . Each volume was primarily drawn on coronal slices, with sagittal and horizontal slices used to confirm regional boundaries. All structures were delineated by one investigator (A.S.) and checked by two others , to ensure protocol conformity.The 7 T2 MRIs in the Atlas group were manually labeled into 27 VOIs, each in native space. The MRI image and its corresponding label volume constitute one atlas of the multi-atlas dataset. The list of these 27 structures, following the nomenclature of the Paxinos atlas, is shown in A maximum probability atlas was created by decision fusion of all 7 individual label volumes in the template space . Individad-hoc maximum probability atlas created by propagation-fusion of the individual atlas dataset in the space of the MRI to be segmented (PF method).Two automated multi-atlas segmentation methods were implemented, one using the maximum probability atlas, (MP method) and the other an The MP method consisted in 1/computing the non-linear registration of an individual MRI on the MRI template; 2/computing the inverse transformation field from template space to individual space; and 3/applying the inverse transformation to the maximum probability atlas and resampling it using the nearest-neighbor (NN) interpolation method to preserve label values .The PF method consisted in propagating and fusing the multi-atlas dataset directly in the native MRI space of the subject to be segmented (target), by 1/computing the non-linear registration of each individual MRI from the atlas dataset, onto the target MRI; 2/using the transformations to resample (NN) the label volumes of the atlas dataset in the target space, and 3/performing label fusion of the individual registered label volumes using maximum probability rules .morphometric and functional evaluation.The performance of the multi-atlas segmentation methods was assessed at two levels: i.e., on the five additional rats of the Test group. Brain structures were manually delineated on the individual MRIs with respect to the protocol, this time by different investigators . All 27 structures were drawn. Accuracy was evaluated in terms of morphometric performance and by measuring functional PET data with automated segmentation.Evaluation was performed on an independent set of co-registered MRI/PET data: Three morphometric indices were used to evaluate the automated segmentation methods. To evaluate volumetric performance, relative volume difference (Eq. 1) and the magnitude  of the relative volume difference (Eq. 2) were computed, quantifying bias in the structure volume extracted by manual versus automated delineation:To evaluate overlap performance, the metric was the Dice similarity index (Eq. 3) Dice ranges from 0 (no overlap), to 1 (complete overlap).manual is the volume of a manually segmented structure, Nautomated that of an automatically delineated structure, and Nautomated∩manual the volume of the overlap between the two.NMorphometric evaluation was performed on the multi-atlas segmentations with the MP and with PF methods. For comparison to more classical segmentation, morphometric evaluation was also performed on regions segmented with an atlas based on a single individual (SA method), instead of the maximum probability atlas in the MP method, using manual delineation of the 65-slice brain MRI.Activity extraction was compared between manually delineated and automatically segmented structures with four different methods: with a single atlas (SA), with a maximum probability atlas, when the individual MRI is available (MP with MRI), or without individual MRI (MP without MRI), and with an atlas obtained by a propagation-fusion (PF).For the MP and SA method, individual PET and MRI acquisitions were registered by computing the rigid spatial transformation of the static PET-FDG image to its MRI. This PET-to-individual-MRI registration was concatenated with the individual MRI-to-template-MRI non-linear transformation. The concatenated transformation was used to transform the single atlas (SA method) or the MP atlas (“MP with MRI” method) in the PET space, where PET-FDG measures were extracted. Alternatively, direct registration between PET-FDG and MRI template was computed. PET-FDG measures were extracted using the resliced MP atlas in the PET space (“MP without MRI”).ad hoc maximum probability atlas was resliced in the PET space to extract regional PET-FDG measures.For the PF method, propagation-fusion of the multi-atlas dataset was performed with the individual MRI as target, and the 18F]FDG activities were converted to standardized uptake values (SUVs). Regression parameters were computed between manual extraction in the individual MRI space versus SA, MP (with or without MRI) and PF methods.Regional FDG PET regional measurement (SUVs) with MP and PF was compared with measurement of manually delineated regions. Regressions, shown in 2 = 0.641; p<0.0001) between single atlas and manual SUVs, and excellent correlations between multi-atlas and manual SUVs  despite slight (non-significant) scatter  with MP and less than 40 minutes with PF. These times are to be compared with the dozens of hours needed for manual delineation.A multi-atlas dataset and anatomical MRI template of the Sprague-Dawley rat were created. The accuracy of automated segmentation with the multi-atlas dataset was checked. Regional PET measurement extraction with an automated atlas was also shown to be equivalent to extraction by manual VOI delineation.Automated segmentation with a multi-atlas dataset did not induce significant bias in VOI measurement compared to manual delineation. The rat is an animal model widely used for the study of neurological diseases, including degenerative processes that may induce regional volumetric alterations. The present multi-atlas method is an accurate and reproducible means of studying large datasets, since it can be performed automatically in a reasonable computing time. Volumetric measurement was, however, assessed on healthy cases, and the inter-individual variability of the structural database was therefore limited to populations without lesions. Study of pathological cases that may be associated with greater morphological alteration might need more sophisticated non-linear registration methods In the Test group, multi-atlas methods achieved overall performance of more than 0.8 for the Dice overlap index. To our knowledge, no previous performance results for a multi-atlas approach in rat have been reported in the literature; studies in bee Additionally, performance might be increased by more sophisticated fusion rules and atlas selection. The present study finally provided 12 atlases, since the initial multi-atlas dataset of 7 can be completed by the 5 of the Test group. With a dataset of 12 atlases, we would be able to implement a multi-atlas method where decision fusion is not performed with the whole dataset, but by selecting a custom subset of atlases. It has been shown in human studies that selecting atlases with an image-based similarity criterion performs better than a random selection, or than using the whole dataset 2≈1, data not shown). Measures were then extracted using multi-atlas automated delineation in the MRI space. Regression slopes for manually extracted data were close to 1; correlation coefficients were very good, with r2 = 0.996 (MP and PF), the regression slope was close to 1. It was concluded that, in spite of the imperfect overlap between manually and automatically delineated regions, the multi-atlas methods were sufficiently precise to be able to extract PET measurements accurately. The tolerability of the slight mismatch of regions is due to the lower resolution of PET compared to MRI: the small segmentation error visible with the MRI resolution was negligible at the resolution of the PET acquisitions. Our data also shows that using a single individual to create an atlas is largely sub-optimal in comparison to the use of a dataset of multiple atlases that are combined in a maximum probability atlas. The transformations from the individual space and the template space used in the SA and MP methods are the same. They do not explain differences in terms of performance. The SA is clearly biased because it does not contain the inter-individual variability of the structural delineations of a brain region. The MP atlas performs better because it is created from different individuals, and does contain inter-individual variability. Because the MP atlas has a higher signal to noise ratio as a result of averaging, it might be a better target for automated image registration. In addition, multi-atlas methods may better account for subtle differences in slice orientation between different acquisitions which may be present despite the rats being imaged with head restraints.PET regional measurements showed excellent agreement between the manual and automated multi-atlas delineation methods. It was firstly checked that activity extracted by individual manual delineation was completely equivalent in the individual PET, individual MRI and template spaces Click here for additional data file.