Eimeria infected chickens and finally we propose guidelines for optimal annotation strategies.Reliable annotation linking oligonucleotide probes to target genes is essential for functional biological analysis of microarray experiments. We used the IMAD, OligoRAP and sigReannot pipelines to update the annotation for the ARK-Genomics Chicken 20 K array as part of a joined EADGENE/SABRE workshop. In this manuscript we compare their annotation strategies and results. Furthermore, we analyse the effect of differences in updated annotation on functional analysis for an experiment involving IMAD, OligoRAP and sigReannot update both annotation and estimated target specificity. The 3 pipelines can assign oligos to target specificity categories although with varying degrees of resolution. Target specificity is judged based on the amount and type of oligo versus target-gene alignments (hits), which are determined by filter thresholds that users can adjust based on their experimental conditions. Linking oligos to annotation on the other hand is based on rigid rules, which differ between pipelines.For 52.7% of the oligos from a subset selected for in depth comparison all pipelines linked to one or more Ensembl genes with consensus on 44.0%. In 31.0% of the cases none of the pipelines could assign an Ensembl gene to an oligo and for the remaining 16.3% the coverage differed between pipelines. Differences in updated annotation were mainly due to different thresholds for hybridisation potential filtering of oligo versus target-gene alignments and different policies for expanding annotation using indirect links. The differences in updated annotation packages had a significant effect on GO term enrichment analysis with consensus on only 67.2% of the enriched terms.In addition to flexible thresholds to determine target specificity, annotation tools should provide metadata describing the relationships between oligos and the annotation assigned to them. These relationships can then be used to judge the varying degrees of reliability allowing users to fine-tune the balance between reliability and coverage. This is important as it can have a significant effect on functional microarray analysis as exemplified by the lack of consensus on almost one third of the terms found with GO term enrichment analysis based on updated IMAD, OligoRAP or sigReannot annotation. High throughput gene expression experiments using microarrays are based on the principle of hybridising strands of nucleotides to form a duplex. For each gene (the target) a microarray contains many copies of one or more short strands (the probes) in small regions on the array called spots. In a microarray experiment expressed sequences or sequences derived thereof are labelled and allowed to hybridise to the probes making the amount of label at each spot an indicator for the amount of gene expression. Since all spots are processed simultaneously, it is essential that all probes have optimal target specificity under the same experimental conditions. Therefore, optimal microarray design requires 1) a completely sequenced reference genome, 2) complete annotation for this reference genome to know what parts may be expressed and 3) complete knowledge about the natural variation amongst the sampled individuals.Unfortunately there is currently not a single species for which such complete information is available. Although some reference genomes are now close to completion, the recently published first results of the ENCODE project indicate that our knowledge of what is expressed is vastly underestimated . Hence, Previous re-annotation studies have shown that up to half of the probes for popular microarrays can be problematic as they suffer from cross hybridisation, from detecting something else than what they were designed for, or both -7. The sOther evidence that current probe annotation is often suboptimal comes from microarray reproducibility studies. Although reproducibility of modern arrays using the same array platform and version is usually good to very good, reproducibility between different array versions even on the same platform can be very poor -14. Re-aEimeria infected chickens, which was selected as starting material for the joined EADGENE [Summarising, it is important to update the annotation for arrays regularly to improve the reliability of probe-target assignments. Three tools to update oligo annotation for microarrays – IMAD, OligoRAP and sigReannot – are described elsewhere in this issue of BMC Proceedings -18. In t EADGENE  and SABR EADGENE  workshopab initio in silico gene predictions, 3) miRBase micro RNAs and 4) a small set of contributed sequences. Microarray data from an experiment with Eimeria infected chickens and using this array was provided as starting material for the EADGENE/SABRE post-analyses workshop [The microarray used is the ARK-Genomics Chicken 20 K array consisting of 20.460 probes ranging in length from 60 to 75 nucleotides with the majority of the probes 70 nucleotides long . It was workshop . From thworkshop  enrichmeIMAD, OligoRAP and sigReannot were used to update annotation as described elsewhere in this issue -18. At tet al. [Hybridisation filter thresholds were synchronised based on He et al.  where poDifferent custom array annotation packages based on Ensembl Gene IDs were made using the updated annotation provided by the IMAD, OligoRAP and sigReannot pipelines. These custom annotation packages were made with Bioconductor  using thFigure The first step requires the oligo sequences and species of interest as input and aligns the oligos with potential targets. An overview of datasources used for the alignments is provided in Figure Firstly, IMAD ignores strand information and hence might link to annotation derived from features located on the opposite strand of a hit. SigReannot is strand-aware, but can link to annotation from the opposite strand if no annotation was found on the hit strand. Most array platforms only detect a single strand and under normal conditions a gene produces only RNA from a single strand. But there can exceptions like in the case of viral reverse transcriptases, some of which can switch templates resulting in chimeric cDNA molecules . IMAD waSecondly, sigReannot uses UTR/intron extension in case no hits were found on Ensembl transcripts. The latter means that sigReannot searches UniGene  as seconIMAD and OligoRAP also use additional sources for probe-target alignments to increase the coverage of annotated probes. In addition to Ensembl transcripts IMAD aligns probes with UniGene  and DFCIThe second step is to filter the hits based on the quality of the alignments, which relates to the hybridisation potential of a hit. All three pipelines can filter alignments on the percentage of sequence identity. Low quality hits that do not pass this filter, but which do contain small stretches of uninterrupted matches might still contribute to signal on a microarray. Therefore, OligoRAP and sigReannot feature a second filter for the minimum size of what is called the longest contiguous stretch or continuous block, respectively. Finally, OligoRAP has a third filter for the maximum total amount of mismatches. When the probes are not all equally long this filter will produce different results as compared to the percentage identity filter.In contrast to sigReannot and IMAD, OligoRAP applies the filter step not immediately after aligning oligos with targets, but after all annotation is retrieved instead (after step 4). This allows OligoRAP to check if two or more short hits were derived from intron-separated exons of the same gene. If such hits are found they are merged into a larger hit, which is necessary for OligoRAP, because it aligns with reference genomes as compared to IMAD and sigReannot, which only align with transcripts.Based on the amount and type of hits oligos can be assigned to target specificity classes (TSCs). An overview of how TSCs overlap or differ between the 3 pipelines is given in Figure OligoRAP and sigReannot use comparable TSCs in case there was only one HQ hit (TSC O1 & S1), there were multiple LQ hits (TSC O5 & S5), or there were no hits at all (TSC O6 & S6).When there was only 1 LQ hit, OligoRAP puts these oligos in a single TSC (O2), but sigReannot differentiates between LQ hits with longest contiguous stretches of 30 nucleotides and more (S3) and with stretches of less than 30 nucleotides (S4). The latter TSC contains gene-specific oligos, which are less reliable for detecting lowly expressed genes, because they have the worst signal to noise ratio. By providing an extra TSC for these oligos, users can choose to drop them from further analysis or at least can see quickly they are less reliable. OligoRAP handles this problem differently by allowing users to specify multiple combinations of filter thresholds per run. This allows them to analyse for example the effect of more lenient or stricter thresholds for HQ and/or LQ hits and covers all TSCs instead of just the oligos with only one LQ hit. Analysing different combinations of filter thresholds is also possible with IMAD and sigReannot, but this a bit more work as it requires a user to run the pipeline with the most lenient hybridisation potential filter thresholds followed by post-processing of the results to generate results for more stringent thresholds.In the case of multiple HQ hits or a mix of HQ and LQ hits sigReannot and OligoRAP classify them differently. SigReannot differentiates between cases with one HQ hit accompanied with one or more LQ hits (TSC S2) and cases with multiple HQ hits with or without LQ hits (TSC S7). OligoRAP on the other hand differentiates between multiple HQ hits (TSC O3) and a mix of HQ and LQ hits (TSC O4). The reason sigReannot differentiates between S2 and S7 while OligoRAP assigns such oligos all to O4 is a difference in annotation retrieval policy (see below). TSC O3 is interesting, because in theory these oligos target shared domains or different highly similar genes. Therefore these oligos could still be informative as such genes are usually involved in similar biological processes just like different splice variants derived from the same gene. In practice however many of the oligos in TSC O3 have multiple HQ hits due to redundancy and this is usually the result of assembly and/or annotation problems. Either way it makes sense to differentiate between these oligos and ones that target a mix of HQ and LQ hits as the latter can suffer from cross-hybridisation with transcripts from highly dissimilar targets and hence are not informative.Steps three and four consist of annotation retrieval directly from the alignments or indirectly from previously fetched annotation, respectively. There are many differences in annotation features retrieved by the different pipelines, but all pipelines provide links to Ensembl gene IDs, Ensembl Transcripts IDs, UniGene cluster accessions and GO terms IDs derived from Ensembl genes Figure . The larFinally, the fifth step involves formatting and storing the results in various ways. SigReannot's annotation is provided as collection of tab-delimited flat files. IMAD on the other hand uses a MySQL database to store its results and OligoRAP's native output format is BioMoby  XML, butA subset of 791 oligos was selected from the experimental data provided for the workshop to assess the effect of the different annotation strategies on coverage. These oligos were selected, because they showed differential gene expression signals. Hence these probes clearly bind transcripts and any orphan oligos in the updated annotation produced by sigReannot, OligoRAP and IMAD indicate false negatives due to incomplete data sources, incomplete annotation strategies or both. The focus for this comparison is on Ensembl gene ID assignments as all three pipelines provide these and hence they can be easily compared. Figure In case an oligo was not linked to any Ensembl genes by any of the pipelines, they clearly all agree, but in case two or more pipelines link to Ensembl genes, that does not necessarily mean they link to the same Ensembl genes for the corresponding oligos. Therefore, the consensus between the pipelines in linking oligos to Ensembl genes was determined Figure . For 44.In case the annotation pipelines did not agree on the Ensembl genes linked to an oligo the reason for this lack of consensus was determined Figure . MultiplIn the vast majority of the remaining cases where consensus is lacking, the pipelines initially find the same hits, but judge them differently when deciding whether to link to Ensembl genes based on these hits or not. In 88 and 66 cases oligos are linked to Ensembl genes located on the opposite strand of the hit with IMAD and sigReannot, respectively. The difference is the result of sigReannot not linking to annotation from the opposite strand if there was also annotation on the strand of the hit. Furthermore, sigReannot only takes annotation from the opposite strand into account for HQ hits and hence ignores such annotation for LQ hits. So sigReannot is a bit more conservative in linking to annotation from the opposite strand.OligoRAP only links a hit to annotation if there is (near) perfect overlap between the hit  and the annotation . For IMAD and sigReannot this does not apply as they only align the oligos with transcripts. In case a hit extended beyond the borders of a transcript on the genome, IMAD and sigReannot will find a shorter hit covering only the part that overlaps with the transcript. This results in 24 oligos with extra links to Ensembl genes with IMAD and 17 with sigReannot as compared to OligoRAP. The difference between IMAD and sigReannot is the result of partial overlap combined with lower thresholds for IMAD either because of the lack of a contiguous stretch filter in IMAD (2 cases) or because the annotation was derived from the opposite strand and the hit didn't pass sigReannot's HQ hit thresholds (5 cases).SigReannot's UTR/intron extension feature generates additional links to Ensembl genes for 16 oligos with hits in the vicinity of Ensembl transcripts. IMAD and OligoRAP cannot link to annotation located in the vicinity of a hit on the genome and this explains their absence.If a BLAST result contains overlapping HSPs these were all ignored by IMAD resulting in 7 oligos where links to Ensembl genes are missing as compared to OligoRAP and sigReannot. Further inspection of these 7 probes revealed they contained repeats and IMAD has been adjusted to include hits from overlapping HSPs.Finally, the "other" leftover category contains 9 rare cases. In one of these IMAD missed a hit, because it uses BLAST ,37 with It must be noted that IMAD and sigReannot normally provide maximally a single link to an Ensembl gene per oligo. In case there are multiple hits for an oligo these pipelines will try to find a best one and if this fails not link to Ensembl at all. For this workshop the IMAD and sigReannot teams provided additional data for oligos with multiple hits, so they could all be taken into account and compared, but would users compare standard IMAD and sigReannot data, they might find additional differences due to different prioritisation of hits to find the best one.In most cases the oligos with multiple hits are non-specific, but further investigation revealed 9 extreme cases of oligos with numerous hits (up to 200) on transcripts representing large gene families or sharing domains such as genes coding for MHC proteins, olfactory proteins, homeobox proteins, protein kinases and potassium voltage-gated channel proteins. Although it was clearly not possible to assign a best hit in these cases linking the oligo to the gene family or shared domain could still be highly informative despite the lower resolution.et al. [GO term enrichment analysis was chosen as an example to investigate the results of differences in updated annotation on functional microarray analysis. For this analysis all probes of the ARK-Genomics 20 K chicken array were taken into account, annotation was updated with IMAD, OligoRAP & sigReannot and enrichment of GO terms in the lists of significantly up- or down regulated genes was performed as described by Haisheng et al. . Three cIMAD only differentiates between oligos with 1 hit, multiple hits or no hits at all and uses a single hybridisation potential filter for sequence identity. This is less advanced than OligoRAP and sigReannot, which differentiate between LQ and HQ hits and introduce a second hybridisation potential filter for short contiguous stretches of matching nucleotides. Despite these differences, basically all three pipelines can divide the oligos into several TSCs giving users an indication of the target specificity of the oligos. Furthermore, depending on experimental conditions, users can customise the parameters for the hybridisation potential filters.After the pipelines have aligned oligos with potential targets they have to decide whether or not to link to certain annotation based on these alignments and this is where they differ the most. Should the pipelines be very conservative and link only to annotation derived from other sequences that (near) perfectly overlap the alignment of the oligo with the potential target, like OligoRAP does? Or should the annotation strategy be more lenient and include annotation from indirect links like sigReannot does when it uses UTR/intron extension to link indirectly to Ensembl via UniGene? The question basically boils down to whether to prefer reliability over coverage or the other way around. After some discussion during the workshop the biologists present decided they couldn't choose between optimal coverage and optimal reliability. Instead they would prefer to have as much annotation as possible and have metadata attached to the annotation indicating the reliability of the link between the oligo and for example an Ensembl gene. Similar to the target specificity categories one can think of a few annotation link reliability categories that would allow the biologists to filter their results in downstream analysis and see the effect of in- or excluding less reliable annotation in addition to the effect of in- or excluding potential non-specific oligos. We propose the following categories:1) Direct sequence-based links: annotation was derived from alignment of the oligo with a target sequence.2) Indirect linksa) Sequence-based and with (near) perfect overlap of the oligo-target alignment with the alignment of the target with the other sequence from which the annotation is derived.b) Sequence-based and with partial overlap of the oligo-target alignment with the alignment of the target with the other sequence from which the annotation is derived.c) Sequence-based and without any overlap of the oligo-target alignment with the alignment of the target with the other sequence from which the annotation is derived.i) Oligo-target alignment is located up- or downstream in the vicinity of the gene from which the annotation is derived.ii) Oligo-target alignment is located in an intron of the gene from which the annotation is derived.iii) Oligo-target alignment is located on the opposite strand of the gene from which the annotation is derived.d) Non sequence-based links. For example in the case of expanding annotation using text mining.e) Non gene-specific link. For example to a gene family or shared domain.These categories can be easily expanded where necessary. For category 2 cii one could flag for example whether there was other sequence-based evidence that makes the link more reliable. This would be the case if an oligo aligns with an intron and if there are ESTs that align with both the gene's exons and the intron suggesting the gene model was too conservative and intron retention splice variants do exist.Approximately four years after the design of the ARK-Genomics 20 K chicken array almost one third of the probes could no longer or still not be linked to high quality annotation in the form of a link to an Ensembl gene with neither IMAD nor OligoRAP nor sigReannot. This indicates that keeping annotation as well as target specificity up-to-date is important to make most of microarray experiments.IMAD, OligoRAP and sigReannot can assign oligos to target specificity classes (TSCs) although with different levels of resolution. These TSCs are based on the amount of target each oligo hits and users can specify thresholds for hybridisation potential filter used to determine the impact of these hits. Thereby the hybridisation potential filters combined with the TSCs give users the flexibility to adjust the target specificity estimates to their experimental conditions. In addition it allows them to play safe by discarding potential cross-hybridising probes or live on the edge to get higher annotation coverage. In contrast to target specificity users have no control over the annotation that is fetched based on the hits of the oligos with potential targets. Fetching annotation from indirect relationships between oligos and potential targets can help to boost coverage, but will also result in varying levels of reliability of the updated annotation. Not only have users currently no control over which annotation is retrieved, they currently also cannot see the difference between annotation from more reliable direct links and from less reliable indirect links. Based on the feedback from the EADGENE/SABRE post-analysis workshop we therefore suggest annotation link reliability categories be added to indicate the type of relationship between oligos and their annotation. Adding such indicators for the reliability of the annotation will be an important step in the future development of IMAD, OligoRAP and sigReannot and allow users to fine tune the balance between reliability and coverage. This is important as it can have a significant effect on functional analysis of microarray data as exemplified by the lack of consensus on almost one third of the terms found with GO term enrichment analysis using updated annotation generated with IMAD, OligoRAP and sigReannot.Links to supplemental files with annotation as used for the workshop as well as presentations as presented at the workshop are available from the EADGENE portal:The authors declare that they have no competing interests.PBTN generated OligoRAP annotation, PC generated sigReannot annotation and MW generated IMAD annotation. PBTN, PC, MW, DP & CK analysed the generated annotation and compared the different annotation strategies. HN studied the effect of different pipelines and different filter thresholds on GO term over-representation analysis. JAML, MAMG, MW and CK secured funding and managed the project. PBTN drafted the manuscript, which was improved with the help of all other authors. All authors read and approved the final manuscript.


Projections from hippocampal CA1-subiculum (CA1/SB) areas to the prefrontal cortex (PFC), which are involved in memory and learning processes, produce long term synaptic plasticity in PFC neurons. We examined modifying effects of these projections on nociceptive responses recorded in the prelimbic and cingulate areas of the PFC.Extracellular unit discharges evoked by mechanical noxious stimulation delivered to the rat-tail and field potentials evoked by a single stimulus pulse delivered to CA1/SB were recorded in the PFC. High frequency stimulation  delivered to CA1/SB, which produced long-term potentiation (LTP) of field potentials, induced long-term enhancement (LTE) of nociceptive responses in 78% of cases, while, conversely, in 22% responses decreased . These neurons were scattered throughout the cingulate and prelimbic areas. The results obtained for field potentials and nociceptive discharges suggest that CA1/SB-PFC pathways can produce heterosynaptic potentiation in PFC neurons. HFS had no effects on Fos expression in the cingulated cortex. Low frequency stimulation  delivered to the CA1/SB induced LTD of nociceptive discharges in all cases. After recovery from LTD, HFS delivered to CA1/SB had the opposite effect, inducing LTE of nociceptive responses in the same neuron. The bidirectional type of plasticity was evident in these nociceptive responses, as in the homosynaptic plasticity reported previously. Neurons inducing LTD are found mainly in the prelimbic area, in which Fos expression was also shown to be inhibited by LFS. The electrophysiological results closely paralleled those of immunostaining. Our results indicate that CA1/SB-PFC pathways inhibit excitatory pyramidal cell activities in prelimbic areas.Pressure stimulation (300 g) applied to the rat-tail induced nociceptive responses in the cingulate and prelimbic areas of the PFC, which receives direct pathways from CA1/SB. HFS and LFS delivered to the CA1/SB induced long-term plasticity of nociceptive responses. Thus, CA1/SB-PFC projections modulate the nociceptive responses of PFC neurons. The two segregated central pathways for sensory-discriminative and affective dimensions of pain have been examined in human brain imaging studies , which iCA1 pyramidal cells in the hippocampus (HP) receive pain information from peripheral nociceptors . In a raThe projections from the HP to the PFC were established by anatomical ,14 and pWe analyzed the effects of CA1/SB inputs into the prelimbic and cingulate areas of the PFC on nociceptive responses evoked by peripheral mechanical noxious stimulation. HFS/LFS delivered to the CA1/SB induced LTP/LTD-like changes in nociceptive responses recorded in the PFC, suggesting the HP-PFC pathway to be involved in affectional memory in pain processing.Adult male Wistar rats  were used in all experiments. The rats were housed under controlled temperature (25°C) and humidity (40 - 50%) conditions with a 12-h light/dark cycle, and had free access to food and water. Experiments conformed to guidelines issued by the National Institutes of Health for Laboratory Animals and all procedures were approved by the Animal Experimental Committee of Tokyo Women's Medical University. Efforts were made to minimize the number of animals used and their suffering. All rats were anesthetized with a single injection of urethane  and mounted in a stereotaxic instrument . Body temperature was maintained at 37 - 38°C using a chemical thermo-mat.Recording electrodes  were placed in the cingulate or prelimbic areas of the PFC , equipped with a probe with a circular contact area and a 1 mm in diameter tip. Mechanical stimuli were delivered every 90 s at constant force with a feedback system. Stimulus intensities used in this experiment were 300 - 500 gf with a 0.1 s rising (and decreasing) time to maximum force and a 2 s hold time. The stimulus condition applied to the tail evoked c-fiber activities in peripheral nerves .In these experiments, a monopolar stainless steel stimulus electrode  was lowered into the dorsal portion of the CA1/SB area . At the end of each experiment, the animals were perfused with normal saline and 4% paraformaldehyde. After overnight post-fixation, the brains were sectioned (50 μm) and stained with hematoxylin-eosin solution for examination of the recording and stimulus sites under light microscopy.2 areas in the cingulate and prelimbic areas were counted in 15 slices for each animal.We counted the number of Fos-positive cells in the PFC  with HFSSignificant differences in discharges evoked by mechanical stimuli were assessed with the nonparametric paired-test (Wilcoxon) to compare pre- and post-stimulation values. Data are expressed as means ± standard errors (S.E.). Results for the numbers of Fos positive cells were statistically analyzed with the Mann-Whitney test (untreated group versus HFS/LFS group). A probability level of < 0.05 was considered significant.Noxious mechanical stimulation delivered to peripheral tissue elicited unit discharges and the duration of these responses reportedly reflects stimulus intensity . We measLFS delivered to the CA1/SB induced LTD of nociceptive responses  and 20.4 ± 1.0 (right side) in the cingulate area and 19.7 ± 0.9 (left side) and 21.9 ± 1.3 (right side) in the prelimbic area Fig . There wNociceptive information from peripheral tissue mainly projected to the superficial layers of the cingulate and prelimbic areas , while sNMDA mediated plasticity in the prefrontal cortex was recently reviewed . NMDA reLFS delivered to HP/SB induced LTD in nociceptive responses to peripheral noxious stimuli. LFS  produces LTD mediated by the glutamate receptor, NMDA  and mGulLFS significantly decreased Fos expression in cells in the ipsilateral prelimbic area. The histological data were consistent with the areas in which LTD was recorded in the electrophysiological experiments. LFS delivered to HP/SB may inhibit excitation of prelimbic pyramidal cells via HP-PFC pathways. Thus, LFS decreased Fos expression and induced LTD of nociceptive responses.The PFC is the center of the affectional dimension of pain and involves memories of fear, which are formed by pain experiences. Strong sensory information from peripheral nerves, such as the effects of amputation-induced LTP on synapses receiving information from sensory nerves, may be the cause of phantom pain . ConnectAll three authors participated in the preparation of this manuscript. and approved the final manuscript. The individual contributions of three authors to the manuscript are below.H Nakamura carried out the electrophysiological studies and statistical analysis.Y Katayama carried out the immunostaining and statistical analysis of Fos expression.Y Kawakami conceived the study and coordinated all experiments.


Hippocampus abdominalis, has an exceptionally highly developed form of male parental care, with female-female competition and male mate choice.Both natural and sexual selection are thought to influence genetic diversity, but the study of the relative importance of these two factors on ecologically-relevant traits has traditionally focused on species with conventional sex-roles, with male-male competition and female-based mate choice. With its high variability and significance in both immune function and olfactory-mediated mate choice, the major histocompatibility complex (MHC/MH) is an ideal system in which to evaluate the relative contributions of these two selective forces to genetic diversity. Intrasexual competition and mate choice are both reversed in sex-role reversed species, and sex-related differences in the detection and use of MH-odor cues are expected to influence the intensity of sexual selection in such species. The seahorse, Here, we demonstrate that the sex-role reversed seahorse has a single MH class II beta-chain gene and that the diversity of the seahorse MHIIβ locus and its pattern of variation are comparable to those detected in species with conventional sex roles. Despite the presence of only a single gene copy, intralocus MHIIβ allelic diversity in this species exceeds that observed in species with multiple copies of this locus. The MHIIβ locus of the seahorse exhibits a novel expression domain in the male brood pouch.The high variation found at the seahorse MHIIβ gene indicates that sex-role reversed species are capable of maintaining the high MHC diversity typical in most vertebrates.Whether such species have evolved the capacity to use MH-odor cues during mate choice is presently being investigated using mate choice experiments. If this possibility can be rejected, such systems would offer an exceptional opportunity to study the effects of natural selection in isolation, providing powerful comparative models for understanding the relative importance of selective factors in shaping patterns of genetic variation. The impact of natural and sexual selection on genetic diversity has been intensively studied in both natural and captive-bred populations , but theThe hypervariable major histocompatibility complex (MHC/MH) has proven to be a powerful model in which to investigate the importance of natural and sexual selection in shaping genetic diversity -8. The MThe investigation of MHC genes in a diversity of vertebrates indicates that these loci are more diverse than any other gene family . NaturalDespite consistently high levels of variation, there are major differences in the genomic organization of MHC genes in different vertebrate groups. While these loci are physically linked in mammals, class I and II genes are unlinked in bony fishes (class Actinopterygii) ,23. Due While previous studies on teleosts have shown that both natural and sexual selection structure MH allelic diversity in species with conventional female-based mate choice ,30,31, nHippocampus abdominalis, have found evidence of female-female competition and male mate choice, suggesting that natural populations of this species are sex-role reversed [The teleost family Syngnathidae (seahorses and pipefish) is a well-suited model system to study questions concerning the relationship between sex roles and MH diversity. Both conventional and sex-role reversed species exist in the family and sex-role reversal has evolved several times independently in this group . Studiesreversed .Here, we characterize MH-variation in wild-caught and captive-bred individuals of sex-role reversed populations of the potbellied seahorse, a species with a highly developed form of male parental care. Genome sequencing and transcriptome screening confirm the existence of a single, highly variable copy of the MHIIβ locus in this species, with a pattern of variation identical to that detected in species with conventional sex roles. This pattern of genetic variation has been influenced by a combination of intralocus recombination and positive selection on sites believed to be important for peptide binding. MHIIβ is expressed in brood pouch tissues of male seahorses, suggesting that these molecules may be functionally active during male pregnancy. Our results indicate that sex-role reversed taxa such as the seahorse are capable of maintaining the high MHC diversity typical of vertebrate species with conventional sex roles.Hippocampus kuda: e-value = 0.0, Hippocampus sp.: e-value = 2e-100, Monopterus albus: e-value = 2e-35, Archoplites interruptus: e-value = 1e-33, Tetraodon nigroviridis: e-value = 1e-33). The structure of MHIIβ in the seahorse is similar to that in other vertebrates, with 6 exons separated by 5 introns of varying length  located in introns 2 and 4 , but none of these values remained significant after correcting for multiple comparisons . The 17 alleles include 25 polymorphic nucleotide sites and a total of 17 amino acid differences  were also detected in the captive-bred population. The nucleotide diversity π of the seahorse MHII β1-domain is 0.034. The dataset used for subsequent analyses contains 270 bp of exon 2 , after omitting exon-spanning codons at the 5' and 3' ends of the exon .Sequencing of the highly-variable peptide binding region of the seahorse MHIIβ locus identified a total of 17 s Figure . 86% of Only 2 of the 25 nucleotide substitutions detected in exon 2 of the seahorse are synonymous, leading to a dN/dS ratio of 3.7 . A network without these recombinant alleles is qualitatively similar to the full network, but the placement of Hiab-DAB-E2*09 shifts in the pruned dataset, reflecting its high level of divergence from the central haplotypes . These culeatus , Oncorhyviatilis  and Poecticulata ). As malticulata ,33, the ticulata ,20, and ticulata , but theH. abdominalis: 17 alleles in 101 individuals, Oncorhynchus gilae gilae: 5/142, O. tshawytscha: 12/144, Salmo trutta: 24/180, O. mykiss: 88/423), but exhibits fewer polymorphic sites  than that found in salmonids  [H. abdominalis and salmonids show comparable nucleotide diversities in the PBR-containing β1-domain of exon 2  -43. H. a = 0.054 ).Gasterosteus aculeatus), an important model system for the study of teleost MH evolution, are thought to carry at least 4 copies of MHIIβ [Poecilia reticulata, a species with at least 2 MHIIβ loci, recovered 18 exon 2 alleles in 56 individuals  [Poecilia formosa [Perca fluviatilis [As interlocus gene conversion is thought to contribute to the diversity of gene families , one migof MHIIβ ,26,45. Acus ≤ 9) . This pa formosa  and Percviatilis , with 9 viatilis ,48 and bviatilis . These fPoecilia spp.  and per, illustH. abdominalis), a species with an exceptionally well-developed form of paternal care and male mate choice. The sex-role reversed H. abdominalis exhibits levels of MHIIβ diversity similar to that detected in species with conventional sex roles. This species has a single functional MH class II beta-chain gene that is expressed in the male brood pouch, suggesting that this gene may be immunologically active in these tissues. The pattern of MHIIβ genetic diversity in the seahorse has been influenced by positive selection and recombination, and intralocus genetic diversity in this species exceeds that present in species carrying multiple copies of this gene. Mating experiments are currently being used to determine whether MH-odor cues are used in mate choice decisions in H. abdominalis, data which should help to shed light on the relative roles of natural and sexual selection in generating the high levels of MHIIβ diversity found in the seahorse.We provide the first data on the pattern of MH diversity in the seahorse . Sequences were aligned in BioEdit v.7.0.9.1 [Whole genomic DNA was extracted from muscle tissue of a single protocol . To charGenBank:Z077, StizTo amplify MHIIβ, we used long-range PCR under the following conditions: 1× ThermoPol reaction buffer (NEB), 1.2 μM dNTPs, 0.9 μM of each primer, 1.5 U of a 1:20 Pfu DNA polymerase (Promega) and Taq DNA Polymerase (NEB) mixture and approx. 60 ng DNA per 30 μL reaction. PCR running conditions involved an initial denaturation at 92°C for 5 min, followed by 35 cycles of 92°C for 30 sec, 58°C for 30 sec and 68°C for 0.5 - 4 min (depending on product length), with a final extension at 68°C for 5 - 15 min.As the initial primer set provided only a fragment of the MHIIβ locus, genome walking was used to complete the sequence using a protocol modified from the Universal GenomeWalker Kit (Clontech). One μg of high-quality genomic DNA was digested separately with 10 U of the enzymes EcoRV (NEB), PvuII (NEB), StuI (NEB), DraI (NEB), AluI (Promega), HincII (NEB) and Cac8I (NEB) according to the manufacturer's recommendations. Purification of digested DNA and adaptor ligation followed the Clontech protocol. Genome walking was performed using a nested PCR approach with 1× ThermoPol reaction buffer, 1 μM dNTPs, 0.4 μM AP1 primer, 0.4 μM gene-specific primer 1, 1 U Taq DNA polymerase (NEB) and 1 μL of the DNA-adaptor-library in a 20 μL reaction volume for the first round PCR. The nested PCR was performed using the same protocol, but with the AP2 primer and a nested gene-specific primer along with 1 μL of a 1:50 dilution of the initial PCR product. Cycling conditions were identical in both PCRs, with 2 min at 92°C, 30 cycles of 30 sec at 92°C, 30 sec at 57°/60°/63°C and 3 min at 68°C.PCR products were purified for sequencing using either a MultiScreen PCR filter plate (Millipore), gel-purification with the Wizard SV Gel and PCR Clean-Up System (Promega), or via cloning with a Topo TA Cloning Kit (Invitrogen) following the manufacturers' recommendations. 10-20 positive colonies per plate were picked into 25 μL of ddH20, directly PCR-amplified and sequenced. Cloned products were compared to direct sequences generated with several different primer combinations, in order to identify allelic phase and to identify any cloning-mediated PCR artifacts. Purified PCR products were prepared for sequencing by adding 1 μL Big Dye v3.1 Terminator Cycle Sequencing mixture (Applied Biosystems) and 1 μL primer to 2-8 μl of purified product in a 10 μL volume. Cycling conditions were 30 cycles of 10 sec at 96°C, 5 sec at 50°C and 4 min at 60°C. Ethanol-purified products were sequenced on an ABI 3730 automated sequencer (Applied Biosystems).® Reagent (Invitrogen) according to the manufacturers' recommendations. One μg of purified RNA was digested with 9 μL of DNase I (Promega) and reverse-transcribed into cDNA with 1 μL ImProm II Reverse Transcriptase (Promega) using 2 μL of a 500 μg/μL solution of a dT-adaptor primer (TAGGAATTCTCGAGCGGCCGCTTTTTTTTTTTT) in 25 μL volume. The program for the RT-PCR followed the manufacturer's recommendations (Promega). 3 μL of a 1:2 dilution of Millipore-purified cDNA was used as template in a PCR reaction with MHIIb-E1F2 and MHIIb-E6R under the standard PCR conditions outlined above.To determine whether MHIIβ sequences obtained from genomic DNA represent functional alleles, we amplified and sequenced a partial MHIIβ cDNA sequence (exon 2 - 5) from liver, muscle and pouch tissue of a reproductively mature non-pregnant male seahorse. RNA was extracted using TRIZOLH. abdominalis possesses a single functional MHIIβ gene (see below). To further explore this pattern, we screened cDNA libraries of seahorse pouch and reference tissues from pregnant and non-pregnant individuals for the presence of MH genes using 454 sequencing. Briefly, both normalized and unnormalized cDNA libraries prepared from purified total RNA derived from the pouch tissues of a single pregnant and non-pregnant seahorse, together with a pool of normalized reference tissues from the pregnant individual , were individually MID-tagged with a unique sequence identifier. MID-tagged libraries were sequenced using GS FLX Titanium Chemistry (Roche), following the manufacturer's recommendations. A full plate of 454 sequencing yielded a total of 850 K filtered reads (average read length 230 bp), 92% of which could be assembled into 38 K contigs. The full results of this transcriptome screen will be described in detail elsewhere .Genomic DNA and cDNA sequencing indicate that In order to investigate the hypervariable PBR of MHIIβ, complete exon 2 sequences were amplified in an additional 100 individuals as part of a larger study investigating MH-based mate choice preferences in the seahorse. Seahorses are listed under Appendix II of the United Nations Convention on the International Trade in Endangered Species (CITES), and the majority of the samples included here thus originate from a captive-bred population derived from individuals collected from several sex-role reversed Tasmanian populations. The seahorses in this captive-bred population are held in large communal breeding tanks  with 50 males and 50 females per tank, allowing free mate choice . This population is genetically diverse  and an individual-based assignment test indicates the existence of a single Tasmanian population of captive-bred and wild-caught individuals (Structure: Pr(K = 1) = 1; see Additional file We obtained exon 2 sequences from 47 F1 individuals from 5 families (n = 8-13 per family), to investigate whether MH alleles segregate in a Mendelian fashion. This approach demonstrates the mode of inheritance of these loci and provides a means to evaluate the reliability of sequence profiles generated for this fragment of the MHIIβ gene, through parent-offspring comparisons.Hiab-DAB-E2*01-17, following standard terminology [Sequence data were assembled using Sequencing Analysis 5.2 (Applied Biosystems). Sequences were aligned with Muscle v.4.0  and veriminology . MH haplminology , an apprminology . SeqPHASminology .DnaSP v.4.90.1  was useddN and dS were calculated using Mega v.4.0.2  under a RECCO v.0.93  [RECCO is based on a minimal cost solution, in which the relative cost of obtaining a sequence in an alignment from the other sequences by mutation and recombination is evaluated.Recombination in the seahorse exon 2 dataset was tested using the default settings of tations) . The ideAB participated in the design of the study, carried out the laboratory work and data analysis and wrote the manuscript. ABW conceived the study, supervised the laboratory work and data analysis and helped to draft the manuscript. Both authors read and approved the final manuscript.Figure S1: Genetic structure plot.Click here for file


The relationship between weight loss and mortality has important clinical and public health significance but has proved to be complex. Evidence is mixed and particularly limited on the association between weight loss in mid-life and premature death (i.e. before 65 years of age), a small albeit important segment of total mortality. We aimed to study the association between midlife weight change and mortality accounting for health and lifestyle characteristics, and also considering potential bias due to preexisting chronic diseases and smoking status.Longitudinal, population-based, ‘the 1946 British’ birth cohort study.In 2750 men and women, mortality from age 53 through 65 years was analyzed according to categories of measured 10 year weight change between 43 and 53 years. Cox's hazard ratios (HR) were progressively adjusted for socio-demographic, lifestyle and health characteristics.Nearly 20% of participants lost weight and over 50% gained 5 kg or more in midlife. There were 164 deaths. Compared to those who gained between 2 and 5 kg, those who lost 5 kg or more had an increased risk of premature death independently of midlife physical activity, socio-economic circumstances and educational attainment. This association was unaltered when highest weight loss (lost more than 15 Kg) (p = 0.04) and early deaths were excluded (p<0.001), but was no longer significant after adjustment for cardiovascular risk factors and health status .The inverse association between weight loss in midlife and higher risk of premature death may be explained by vascular risk factors and ill health. In consideration of the burden of premature death, closer monitoring of weight loss in mid-life is warranted. Premature death (i.e. before 65 years of age) is a small segment of total mortality but of great clinical and public health importance We used data from a British birth cohort study to test the hypothesis that weight loss over a 10-year period in midlife (43–53 years) would be associated with an increased risk of all-cause premature death between 53 and 65 years. We also investigated whether health conditions , smoking, and body size at age 43 explained any observed associations.Participants provided written informed consent and the Multicentre Research Ethics Committee (MREC) approved the study.http://www.nshd.mrc.ac.uk/data.aspx).Bona fide researchers can apply to access the NSHD data via a standard application procedure  is an ongoing birth cohort study of a socially-stratified sample of 5 362 newborns born in England, Scotland and Wales in one week in March 1946, and followed up 24 times. Height and weight were measured at all study waves to the nearest 0.1 kg with participants in light clothing and no shoes (self-reported at 20 and 26 years). In 1989 and 1999 trained nurses used standardized protocols at home visits to assess health and lifestyle characteristics. Cohort members were flagged for death on the National Health Service (NHS) Central Register. The start of mortality follow-up was taken as 1999 when cohort members were aged 53 years and ended in March 2011, just before the study members' 65th birthday. Of the 5 362 cohort members 469 had died before age 53 years, 640 had withdrawn from the study, 580 lived abroad, and 638 were untraced or non-responders. Of the remaining 3 035, we excluded those who were not weighed and measured at both 43 and 53 years (n = 274) and those who did not have mortality data available (n = 11) leaving 2 750 for analysis. A further 182 were excluded from complete-case analyses due to missing covariate information.The exposure variable was 10 year weight change calculated by subtracting weight at age 43 from weight at age 53 years. We derived a categorized variable for weight change using the following groups: We classified educational attainment achieved by the age of 26 years according to the Burnham scale into: 2. A priori power calculations (at 90% and 5% significance) confirmed that a hazard ratio (HR) of 1.03 per kg of weight change could be detected assuming a linear relationship. Weight change predicted mortality similarly in men and women , so results are presented for men and women together adjusted for sex.We compared participants' characteristics across weight change categories using ANOVA and χ2) at 43 years, and by smoking status (yes/no) at the beginning of the weight loss period (43 years) to formally test whether obesity and smoking status modified the association of weight change with mortality. We also conducted separate analyses by subgroup where evidence of an interaction was observed. Finally, because weight change is likely related to body size, in additional models we investigated the relationship between BMI  at 43 years and mortality controlling for sex, and weight change. A similar model was fitted replacing BMI at 43 years with BMI at 53 years. We conducted the statistical analyses using STATA software, version 12.0 .Survival across weight change categories was first assessed by graphical inspection of Kaplan-Meier plot, and a formal test using Schoenfeld residuals showed no violation of the proportionality assumption (P = 0.41). To assess the relationship between weight change and mortality we performed two separate analyses. First, we included weight change as a continuous variable and used restricted cubic spline with three knots to assess the shape of the association with mortality rates. The association with mortality was assessed using the Cox proportional hazards model adjusting for sex and weight at 43 years. The p-value for overall significance of the association was obtained comparing the spline models with the null model using a Wald test . Second, the relationship between weight change and mortality was modeled using the Cox model with weight change in categories (above), initially on the maximum sample with weight change available . The unadjusted model was then refitted on a reduced sample with complete data on all covariates (n = 2 568) to assess potential bias introduced by missing data. Adjustment was then made for socio-demographic, lifestyle and health characteristics in stages to assess confounding. To account for the possibility that existing underlying disease, which results in mortality, may have caused the weight loss, Similar percentages of men and women experienced weight loss, and 11% lost more than 2 kg and 48% gained more than 5 kg between 43 and 53 years. Participants grouped by weight change categories differed across a number of factors . For insThe analysis using spline models suggested that the association between weight change and mortality rates was not linear . There wIn sensitivity analyses, when participants who lost more than 15 kg were excluded (n = 20), the HR for the highest weight loss category (−5 to <−15 Kg) compared with the reference group was only modestly attenuated in a model adjusted for sex and body weight at age 43 years  when compared with the main analysis. Similarly, when early deaths (before age 55 years) (n = 12) were excluded, the HR for the high weight loss group was significant through to model 3 . Also consistent with the main analysis, the HR was considerably attenuated and not significant in the fully adjusted model .In stratified analyses, the increased mortality rate in the highest weight loss group (compared to the reference) was stronger and remained significant after full adjustment (model 4) in non-smokers  but not in smokers at age 43 years  . There was no evidence of an interaction between weight change and obesity status (BMI>30 kg/m2) at 43 years (p = 0.50). Finally, we found that participants who were obese at 43 years  had a higher mortality risk  compared to those who were in the normal weight category  at the same age. This association was independent of their weight change between 43 and 53 years and was hardly attenuated by adjustment for all other covariates. Conversely, those who were obese at 53 years  did not have a significantly higher mortality risk  compared to those who were normal weight (n = 902) in similar models.In this prospective birth cohort study we found that compared to modest weight gain (less than 5kg), weight loss of more than 5 kg in mid-life was associated with a higher risk of mortality before old age . The association was strongly attenuated when cardiovascular risk factors and health status were taken into account.The obesity epidemic is spreading worldwide Our finding that health state strongly attenuates the association between weight loss of more than 5kg and mortality is consistent with a previous study also conducted in a UK population-based sample Our study has limitations. Mortality risks were higher amongst those who were not compared to those who were included in our analysis Overall our findings may reflect illness-related weight loss, In the present study the direct association between weight loss and risk of premature death was largely explained by modifiable risk factors before and treatable clinical conditions after weight loss occurred, and was independent of obesity at 43 years. Together this may suggest that weight loss in midlife as well as obesity warrants monitoring to improve prevention and tailor treatment.


Data sharing is challenging but important for healthcare research. Methods for privacy-preserving data dissemination based on the rigorous differential privacy standard have been developed but they did not consider the characteristics of biomedical data and make full use of the available information. This often results in too much noise in the final outputs. We hypothesized that this situation can be alleviated by leveraging a small portion of open-consented data to improve utility without sacrificing privacy. We developed a hybrid privacy-preserving differentially private support vector machine (SVM) model that uses public data and private data together. Our model leverages the RBF kernel and can handle nonlinearly separable cases. Experiments showed that this approach outperforms two baselines: (1) SVMs that only use public data, and (2) differentially private SVMs that are built from private data. Our method demonstrated very close performance metrics compared to nonprivate SVMs trained on the private data. Data sharing is important for accelerating scientific discoveries, especially when there are not enough local samples to test a hypothesis , 2. Howeϵ. A common mechanism to achieve differential privacy is the Laplace mechanism ) The remainder of the paper is organized as follows. g(Δ). The method approximates the original infinite feature space Ω of g(Δ) with a finite feature space p(ω) of g(Δ). Then add the noise to the weight parameters in the primal form based on the new space p(ω) which degrades the approximation accuracy of Ω. Another problem is that the utility bounds use the same regularization parameter value to compare the private and nonprivate classifiers. They take no consideration into the change of regularization parameter incurred by privacy constraints. Chaudhuri et al.  = (1/2b)ex|/b−|. A Laplace noise has a variance 2b2 with a magnitude of b. The magnitude b of the noise depends on the concept of sensitivity which is defined as follows.A common mechanism to achieve differential privacy is the Laplace mechanism  that addf denote a numeric function, and the sensitivity of f is defined as the maximal L1-norm distance between the outputs of f over the two datasets D and D′ which differ in only one tuple. Formally,Let b = Δf/ϵ. To fulfill ϵ-differential privacy for a numeric function f over D, it is sufficient to publish f(D) + X, where X is drawn from Lap(Δf/ϵ).With the concept of sensitivity, the noise follows a zero-mean Laplace distribution with the magnitude D = { | i ∈ Z+,  1 ≤ i ≤ n}, where xi ∈ Rd denotes the training input points, yi ∈ {1, −1} are the training class labels, and n is the size of training data. Here, d is the dimension of input data and “+1” and “−1” are class labels. A SVM maximizes the geometric margin between two classes of data and minimizes the error from misclassified data points. The primal form of a soft-margin SVM can be written asw is the normal vector to the hyperplane separating two classes of data, C is a regularization parameter that weighs smoothness and errors , and fw(xi) = 〈ϕ(xi), w〉, where ϕ(x) : Rd → RF is a function mapping training data point from their input space Rd to a new F-dimensional feature space RF (F may be infinite). Sometimes we map the training data from their input space to another high-dimensional feature space in order to classify nonlinearly separable data. When F is large or infinite, the innerproducts in feature space RF may be computed efficiently by an explicit representation of the kernel function k = 〈ϕ(x), ϕ(y)〉. For example, k = xTy is a linear kernel function for a linear SVM, and k = exp(−||x−y||22/σ2) is a RBF kernel function, which is translation invariant.SVM is one of the most popular supervised binary classification methods that takes a sample and a predetermined kernel function as input, and outputs a predicted class label for this sample. Consider training data l) = max), we can obtain a dual form SVM written asαi ∈ α, i ∈  is a persample parameter and wj ∈ w, j ∈  is a perfeature weight parameter. The weight vector w can be converted from sample weight vector α via w = ∑i=1nyiαixi in the linear SVM.In this paper, we use a RBF kernel function. Our method can be applied to any translation invariant kernel SVM. With the hinge loss In this section, we first introduce a framework overview and then the technical details of our hybrid SVM method. We assume that all data samples follow the same distribution. Here, we assume that all original data from different data sets follow some unknown joint multivariate distribution and all data tuples are samples from this distribution.ρ = T, ρi ∈ Rd in the mapping function of the approximation form to the RBF kernel. Second, with ρ, we transform the private data from the original sample space to the new 2D-dimensional feature space via the mapping function α in the dual space with the transformed private data and w in the primal space via the linear relationship between α and w in the linear SVM. Finally, draw μ from Lap(λ)D2 where ρ. Then users can transform their test data to the new 2D-dimensional feature space with ρ and classify the transformed data with ρ has no privacy risk because it is retrieved directly from public data. More details about hybrid SVM will be given in the successive subsections.z^(x) in . Then wePrivacy Properties. We present the following theorem showing the privacy property of ϵ-differential privacy.w over a pair of neighbouring datasets is λ in step 4 is set to ϵ-differential privacy which completes the proof.For step 1, no private data is used, and hence step 1 does not impact the privacy guarantee. Due to Corollary 15 in  and the H induced by an infinite dimensional feature mapping with a random z. The random finite-dimensional D i.i.d. vectors ρ1,…, ρD from the Fourier transform of a positive-definite translation-invariant kernel function k, such as the RBF kernel function. Then we can obtain an approximation form z(x)Tz(y) of k using the real-valued mapping function z(x) : Rd → RD defined by the following equation:b1,…, bD are i.i.d. samples drawn from a uniform distribution U. z(x) : Rd → RD maps the data from its original d-dimensional input space to the new D-dimensional feature space. Their approach is based on the fact that the kernel function of a continuous positive-definite translation-invariant kernel is the Fourier transform of a nonnegative measure. The uniform convergence property of the approximation form z(x)Tz(y) to the kernel function k has also been proved in [k refers to the RBF kernel function.Rahimi and Recht  approximroved in . In our x in z(x) and only the vectors ρ1,…, ρD are needed to construct the random finite-dimensional ρ1,…, ρD with an optimization function defined as follows:In our problem setting, since a small amount of public data can be considered as follows:minρ∈RD×z(x)Tz(y) of the kernel function k by deploying the public data to compute the ρ, than randomly sampling ρ from the fourier transform of the kernel function k as shown in [w. Fortunately we can employ the differentially private linear SVM approach in [w after transforming all private data to a new 2D-dimensional feature space using the mapping ρ1,…, ρD as follows:Thus, we can obtain a more accurate approximation form shown in . To guarroach in  to compufined in  with theρ1,…, ρD to approximate the RBF kernel function, we can convert RBF kernel SVM in the d-dimensional input space into the linear SVM in a new 2D-dimensional feature space with D2, where With the vectors ace with , then usIn this section, we experimentally evaluate our hybrid SVM and compare it with one state-of-the-art method, called private SVM and on baseline method. We evaluate the utility of the trained SVM classifier using the AUC metric. Hybrid SVM and private SVM are implemented in MATLAB R2010b, and all experiments were performed on a PC with 3.2 GHz CPU and 8 G RAM.Datasets. We used two open source datasets from the Integrated Public Use Microdata Series ,   the US and Brazil census datasets with 370,000 and 190,000 records collected in the US and Brazil, respectively. One motivation for using these public datasets is that it bears similar attributes  as some medical records, but it is publicly available for testing and comparisons. From each dataset, we selected 40,000 records, with 10,000 records serving as the public data pool. There were 13 attributes in both datasets, namely, age, gender, marital status, education, disability, nationality, working hours per week, number of years residing in the current location, ownership of dwelling, family size, number of children, number of automobiles, and annual income. Among these attributes, marital status is the only categorical attribute containing more than 2 values, that is, single, married, and divorced/widowed. Because SVMs do not handle categorical features by default, we transformed marital status into two binary attributes, is single and is married . With this transformation, our two datasets had 14 dimensions. For each dataset, we randomly extract a subset of original data as a public data pool, from which public data is sampled uniformly, and use the remaining 30000 tuples as the private data.Comparison. We experimentally compared the performance of our hybrid SVM against two approaches, namely, public data baseline and private SVM [vate SVM . The pubMetrics. We used the other attributes to predict the value of annual income by converting annual income into a binary attribute: values higher than a predefined threshold were mapped to 1, and otherwise to −1. Here, we set the predefined threshold as the median value of annual income. The classification accuracy was measured by the AUC (the area under an ROC curve) [ϵ, the dataset dimensionality, and the data cardinality . To vary the data cardinality parameter, we randomly generate subsets of records in the training records set, with the sampling rate varying from 0.1 to 1. For various data dimensionalities with the range being 5, 8, 11, and 14, we select three attribute subsets in the US and Brazil datasets for classification. The first five dimensions include: age, gender, education, family size, and annual income. The second eight dimensions contain the previous five attributes, and additionally nativity, owner of dwelling, and number of automobiles. The third eleven dimensions consist of all the attributes in the second 8 dimensions and is single, is married, and number of children. C curve) . The boxFigures Figures ρ randomly from the Fourier transform of RBF kernel. In contrast, hybrid SVM computes ρ via the public data. This helps improve the accuracy of ρ and leads to less variance.Figures ρ with the public data, since a nonlinear optimization equation needs to be solved. As the other private SVM methods, our hybrid SVM is intended for off-line use, and hence the time is generally acceptable for even 14 dimensional datasets.Finally, We proposed and developed a RBF kernel SVM using a small amount of public data and a large amount of private data to preserve differential privacy with improved utility. In this algorithm, we use public data to compute the parameters in an approximation form of the RBF kernel function and then train private classifiers with linear SVM after converting all private data into a new feature space defined by the approximation form. A limitation of our approach is that we used the L-BFGS method , which i


This work presents results in the field of advanced substrate solutions in order to achieve high crystalline quality group-III nitrides based heterostructures for high frequency and power devices or for sensor applications. With that objective, Low Temperature Co-fired Ceramics has been used, as a non-crystalline substrate. Structures like these have never been developed before, and for economic reasons will represent a groundbreaking material in these fields of Electronic. In this sense, the report presents the characterization through various techniques of three series of specimens where GaN was deposited on this ceramic composite, using different buffer layers, and a singular metal-organic chemical vapor deposition related technique for low temperature deposition. Other single crystalline ceramic-based templates were also utilized as substrate materials, for comparison purposes. However, up to date, large volumes of these materials have not been obtained, and, therefore, bulk III-N substrates with extensive surfaces are not available yet. Traditionally the chosen templates for depositions of nitrides are wafers of sapphire, silicon and silicon carbide. These substrates, however, imply disadvantages related to either a high dielectric constant, high leakage currents or to high economic costs.Group-III nitrides (III-N), especially those related to GaN and AlN, have been strategic semiconducting materials in Power Electronics (PE) for the last 30 years, due to their outstanding temperature stability and dielectric strength combined with a wide bandgap2. Among manifold advantages, the use of low temperature co-fired ceramics (LTCC) as an alternative glass-ceramic substrate enables tailoring of crucial properties, such as permittivity or coefficient of thermal expansion (CTE), thus enhancing the systems figures of merit. It can be argued, though, that this material is not fit for PE applications due to its low thermal conductivity, 2-5 W/mK3, but this issue can be overcome by placing metallic via arrays under the high power device4, which is feasible in the LTCC technology.Nevertheless, it still should be possible to lower the production prices by using composite materials made of ceramic fillers embedded into a glass matrix during a sinter process as a base, which demonstrated to be suitable for high-frequency circuits5. Nevertheless, two main handicaps impede the implementation of III-N growth technologies on LTCC-based substrates by often needed conditions: (i) the inherent, relatively high roughness and porosity of this support lead to low homogeneities in III-N structure and composition; (ii) the often used III-N growth temperatures (over 700 °C) may promote ceramic damage and metal contamination. These are the main reasons why the potential of III-N-on-LTCC has still not been fully exploited in order to obtain the atomically long-range periodic structures that are needed for PE active devices, such as High Electron Mobility Transistors (HEMTs), although the possibilities of a LTCC hybrid technology for passive circuit components in such field have already been explored for over a decade10.In principle, the production of LTCC materials is not cheaper than that of Si itself, but the fact that it can be processed layer by layer while in green state eases the fabrication, by using masks and drills, of inner channels for the allocation of a metallic network acting as electrical connectors, passive circuits and thermal drains. Since this architecture of contacts can be placed prior to the production of the active layers of the devices, this leads to a further reduction of both costs and size, as well as to an improvement of the whole assembly performanceIn this work, we show the results after the first stages of novel proposals to overcome the challenges in III-N/LTCC production and, therefore, the first steps in the production of a revolutionary material for power and high-frequency electronics. In this way, worthy qualities of GaN epilayers were reached by: (i) new recipes for improving surface flatness, chemical affinity and CTE matching between LTCC and III-Ns; (ii) the use of intermediate preparation or buffer layers; (iii) the choice of techniques for the synthesis of III-N crystal layers at temperatures much lower than conventional. The GaN quality progressive improvement, up to the achievement of the best quality so far obtained, to the best of our knowledge, for a III-N layer grown on a LTCC (and similar ceramics) substrate, is reported.Different aspects of the fabricated materials are discussed in this section: surface roughness, layers architecture, topology, chemical composition, and crystallographic structure. A comprehensive view of the studied sets of samples is presented in Table 11, a well-established polishing/lapping12 method was applied to LTCC substrates in samples CT3 and CT4 (not in samples CT1 or CT2) before proceeding to further element deposition. A 20-minutes lapping was applied using B4C as abrasive material followed by a 90-minutes polishing step with 1 µm-size diamond particles. By means of this methodology, the achieved LTCC surface roughness (taking into account the pores) had a root mean square deviation (RMS)13 of Rq = (120 ± 5) nm.Some materials utilized as bulk substrates for the nitride-based heterostructures in this work are of ceramic nature, therefore, surface roughness and porosity are, as previously mentioned, very important issues regarding the achievement of a good crystalline structure for the top layers. If this substrate roughness is not low enough, the first depositions of Ga and N atoms will most probably be inhomogeneous, promoting regions of different crystallographic orientations. Therefore, following the fabrication of the LTCC substrates through a sintering processThe LTCC surface is closed after firing, so the surface roughness is determined by the powder fraction of the ceramic filler  contained into the glass (amorphous). Moreover, the polishing opens the intrinsic pores of the material, which are always present. Size and density of these pores depend on the powder composition of the tape and the sintering conditions. Though the so achieved LTCC surfaces are smoother than their non-treated counterparts, the substrate topography still led to the formation of pits in the material surface because occasionally larger pores are opened by the detachment of the filler particles, promoting a rough surface along the overgrown GaN at these positions. Besides, the LTCC roughness limits the deposition rate and the crystallinity of the nitride, which impedes to continue growing a thicker GaN layer as a roughness relieving solution to this problem. As a first step in the characterization of the materials, the amount and density of pits and surface features for the GaN was observed by optical microscopy, as shown in Fig. 14. Figure In addition to this, other techniques, such as Atomic Force and Electron Microscopies, were used at the University of Cádiz (UCA) in order to obtain a more detailed view of these pits, specifically regarding to their depths. This technique was applied by using NT-SS-I SuperSharp tips from Next-Tip SL  to obtain Atomic Force Microscopy (AFM) images, which were afterwards processed with the Gwyddion softwareSq)13. It has to be taken into account that the values in this table indicate pore to pore measurements, using 1 × 1 µm2 areas. Note that if those areas included the surface pits, which could be the case of carrying out the calculations using AFM maps for 5 × 5 or 10 × 10 µm2 areas, Sq values would increase enormously for the samples using LTCC, while they would not significantly change for those samples using non-porous substrates. For example, when studying a 10 × 10 µm2 AFM maps, Sq values for the GaN surface in samples CT4, ST1, SC1 and SC2 turn out to be 672, 30.0, 5.0 and 7.4 nm, respectively. This is a clear indication than the porosity in GaN layer is mainly produced by the porosity in the substrate. In any case, a clear evolution is observed regarding the decrease of the surface roughness of GaN structures along the successive set of samples and growth processes, being remarkable the fact that the roughness values in samples using LTCC, when the growth process is modified, became close to the ones in heterostructures using other substrates with different growth processes.On a more general application, AFM was used to quantify the roughness of the GaN surfaces. Representative areas of such surfaces (between pores) are shown in Fig. Although all heterostructures present a GaN top layer, the layer thicknesses and stacking sequence differ significantly from one specimen to another, also within the same series. The thicknesses, summarized in Table 2O3 as an intermediate layer (CT1) has the most irregular surface formation, since this Al2O3 is achieved as a conglomerate of non-intentional conic-like features, on top of which the AlN/GaN sequence was grown. Surprisingly enough, the layers on top of the Al2O3 were continuous and quite homogeneous along the whole surface. Such BF-DCTEM images explain the look of optical micrographs in Fig. 2 intermediate layer (sample CT2) generates a more uniform support for the AlN/GaN formation. This is clearly because the used sol-gel silica deposition, with a great capacity for filling cavities and surface planarization, smoothens the surface and suppresses the propagation of the LTCC roughness to upper layers. However, the SiO2 placement approach was not applied to substrate materials in other series, since despite that structural advantage, its CTE value is not adjusted to that of the LTCC, as it happens in the case of the Al2O3. Therefore, the use of an alumina interlayer was considered of more interest in the following series.At a first glance, it is clear that the sample using unpolished CT 700 substrate and Al16.As previously mentioned, the heterostructures fabricated using RF-plasma (CT1-3 and ST1) make use of an AlN buffer layer, in order to obtain a better adjustment between the gallium nitride compound and the support below. Note that AlN was considered the best material for that purpose, due to a high chemical affinity to both underneath alumina and above gallium nitride, a more similar basal lattice parameter to GaN than other templates, and the promotion of two dimensional GaN nucleation. In fact, this material has been employed for more than 30 years as the most suitable buffer layer for the later growth of GaNThe intensity in BF-DCTEM micrographs of an electron-transparent material at certain regions decreases with the atomic density of the structure in the direction of the interacting electron beam. Therefore, the dark, thin GaN shown in the Fig. 17. This structure is closer to a single crystal than those in the previous series, which are pure polycrystals. Clearly, the random orientation of those domains stems from the early stages of the growth. The roughness of this surface is still high enough to avoid a proper matching between the III-N structures during the coalescence process, and a multitude of grains remain differently oriented even along the whole growth process.SEM micrographs in Fig. X-ray diffraction (XRD) was utilized to study the details of the mosaic structures on different substrate materials. Figure 2O3 is not a totally compact film, but presents columnar shapes with embedded elongated voids, as a result of an applied anodic treatment to the aluminum precursor of these layers  nm in the measurement carried out with the 200 µm size probe with a roughness layer of (23.5 ± 0.4) nm and a thickness non-uniformity of only 2.6%. As in the case of CT4, the GaN layer thickness obtained by SE is in good agreement with measurements from TEM images . HAADF images have been obtained using a 1 nm STEM probe and an 8 cm camera length, which corresponds to a 20 mrad inner collecting angle for the HAADF detector. On the other hand, EDX spectra have been obtained in standard-less mode using the same STEM probe.TEM working in scanning mode (STEM) allows the application of a variety of chemical sensitive techniques with a high spatial resolution, such as High Angle Annular Dark Field (HAADF) imaging and Energy Dispersive X-Ray (EDX) spectroscopy. For this, it has to be taken into account that the intensity in HAADF images is proportional to 2O3 layers. Such features, which correspond to locally thinner specimen regions, allow confirming the presence of elongated voids mentioned previously.Figure 2 interlayer in SiCer and SiO2 sol gel in sample CT2), though the corresponding spectra are not shown here. Similarly, these types of spectra were taken for the thick top layer in specimens from the last series, revealing that it was also GaN. As a visual example, an EDX linescan for sample CT4 is presented in this figure, illustrating such affirmations. Again in this case, the presence of both Ga and Al signal at the GaN/Al2O3 interface may be due to surface roughness.These affirmations are also supported by compositional quantitative results. Punctual EDX spectra Fig.  corroborIn the long term, the achievements in this work are directed towards the deposition of monocrystalline GaN acting as active material for transistors for PE and high frequency devices. Therefore, it is imperative to characterize the crystallographic structure of the achieved nitride for each case.20 supports the EDX results about the composition of the top layer.One first approach to this characterization is the use of XRD. The diffractograms in Fig. A HRTEM micrograph for the AlN/GaN bilayer in sample CT1, representative of this structure in samples using RF-plasma for the III-N growth, is presented in Fig. 2O3 . Figure 2 interlayer allows to take a SAED including all the materials in this interphase, as presented in Fig. 2, no peaks related to this intermediate layer appear in this diffractogram. It can be observed that the reflections associated to the silicon {200} and the GaN {0002} and {1The Selected Area Electron Diffraction (SAED) patterns in Fig. GaN heterostructures have been fabricated by a low-temperature MOCVD technique using three sets of substrates and three different approaches regarding the growth temperature, its duration and the type of plasma source. Along this work, an evolution on the thickness and crystalline quality of the GaN-on-LTCC has been perceived. In those materials with unpolished LTCC substrate, nano-poly-crystalline layers of 15 nm of GaN on top of AlN buffer were formed. When used a polished substrate and with the same plasma source than the one in the previous case (RF-plasma), the GaN layer, with an abrupt separation to the AlN underneath, improved in terms of crystallinity, roughness and thickness (up to 70 nm). Remarkably, the improved GaN that was fabricated using a DC-plasma source presents a mosaic microstructure with layers thickness larger than 500 nm, formed by single-crystalline columnar grains randomly misoriented (tilted) among each other, but aligned in the polar direction, or close to it, with respect to the substrate surface. When first deposited, the GaN arranges in cubic fashion predominantly, but tends to form hexagonal structures afterwards.Results on the general structure, composition, topology and crystallinity have been reported. To the best of our knowledge, this work presents the highest crystalline quality obtained to the date for GaN grown on top of porous LTCC materials.25; (b) Sitall, a glass-ceramic bulk material based on the Al2O3-SiO2-MgO-TiO2-CeO2-La2O3 system26; and (c) SiCer, a composite formed by a thin Si single-crystalline wafer, bonded to a CTE adapted LTCC green body during sintering27. These substrates were either bought from commercial suppliers or fabricated at the Technical University of Ilmenau (TU Ilmenau). The studied specimens are collected in Table Three sets of specimens have been studied in this work, each one related to a different type of ceramic or glass-ceramic substrate that was used for the deposition of GaN on top of intermediate and buffer layers. These substrates are: (a) HERATAPE® CT 700 from Heraeus (CT 700), a LTCC substrate2O3 at the facilities of the Technical University of Sofia by voltastatic oxidation (40 V) in 0.3 M oxalic acid solution at 15 °C. The samples were gradually immersed by their Al-face in the electrolyte at a rate from 5 to 10 µm/s to obtain a complete transformation of the previously sputtered aluminium layer into a nanoporous aluminium oxide28. As a source of direct current, a power supply (40 V/5 A), manufactured by Voltcraft® Germany, was utilized. The immersion velocity is defined by the substrate roughness  and the thickness of the initial aluminum layer. Also, in sample CT2, a SiO2 layer produced by sol gel technology is used instead of alumina, being fabricated by the researchers at TU Ilmenau29.The alumina interlayers were produced in the form of nanoporous Al2 intermediate layer utilized for the growth of GaN on SiCer forms by Si ambient oxidation during the chamber-to-chamber specimen transportation process.Nitride semiconducting layers were deposited at Lakehead University by plasma assisted, low temperature MOCVD related technique, using a custom-built reactor with three chambers: a loading lock, a UHV chamber and a residual gas analyzer chamber along with a plasma source for nitrogen species. In fact, the 2 nm-thick SiO30. Therefore, though samples are classified in different groups, attending to the substrate, these three different growth processes have to be, then, also taken into account when comparing the results regarding the achieved GaN layers.Regarding the GaN growth, it has to be taken into account that different approaches were applied for the fabrication of this compound: RF nitrogen plasma at 550 °C (samples CT1 and CT2) and at 540 °C (specimens CT3 and ST1), was used with varying pressures and “Nitrogen to Metal-Organic” precursor ratios in order to obtain a 2D growth of GaN. In the mentioned cases, a first step is applied for creating an AlN buffer layer with growing parameters kept in ways in which 3D growths are achieved . On the other hand, for samples CT4, ST2, SC1 and SC2, a DC plasma at 550 °C, applied a period three times longer than the one used in the second series, was utilized to grow directly GaN (without AlN). More details on the LTCC substrate composition and MOCVD parameters for the three series are given elsewhere+ ions using a Gatan model 691 Precision Ion Polishing System. Specific details on the experimental setup for the different (S)TEM related techniques are indicated in further sections of this work.Following the fabrication of these materials, topographic, structural and compositional characterizations were carried out through the use of techniques related to XRD, AFM, Optical Microscopy and SEM, TEM, STEM microscopies. The equipment employed to apply these techniques consisted, respectively, in a Bruker D8 Advance X-Ray Diffractometer, an AFM Bruker Multimode Nanoscope IIIa operating in tapping mode, a DSX510 Olympus digital microscope, a Field-Effect ZEISS GeminiSEM 500 SEM, a FEI Tecnai F30 TEM (operated at 300 kV), a FEG-2010 Jeol and a FEI TALOS STEM microscopes (both working at 200 kV). In order to carry out (S)TEM characterization, samples were first prepared in cross-section XTEM disposition at UCA and thinned down until electron-transparency using traditional grinding-polishing methods and ion-milled with ArSE was applied at UCA to investigate the topography of macroscopic areas of GaN and intermediate layers. SE measurements were performed in the spectral range between 450 and 950 nm with an automatic rotating analyzer J. A. Woollam V-WASE (variable angle) ellipsometer equipped with an automatic retarder.All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).


These associations were not explained by the effects of maternal depressive symptoms after pregnancy, which both added to and partially mediated the prenatal effects. Maternal depressive symptoms throughout pregnancy are associated with increased ADHD symptomatology in young children. Maternal depressive symptoms after pregnancy add to, but only partially mediate, the prenatal effects. Preventive interventions suited for the pregnancy period may benefit both maternal and offspring mental health.Maternal depressive symptoms during pregnancy have been associated with child behavioural symptoms of attention-deficit/hyperactivity disorder (ADHD) in early childhood. However, it remains unclear if depressive symptoms throughout pregnancy are more harmful to the child than depressive symptoms only during certain times, and if maternal depressive symptoms after pregnancy add to or mediate any prenatal effects. 1,779 mother-child dyads participated in the Prediction and Prevention of Pre-eclampsia and Intrauterine Growth Restriction (PREDO) study. Mothers filled in the Center of Epidemiological Studies Depression Scale biweekly from 12+0–13+6 to 38+0–39+6 weeks+days of gestation or delivery, and the Beck Depression Inventory-II and the Conners’ Hyperactivity Index at the child’s age of 3 to 6 years . Maternal depressive symptoms were highly stable throughout pregnancy, and children of mothers with consistently high depressive symptoms showed higher average levels (mean difference = 0.46 SD units, 95% Confidence Interval [CI] 0.36, 0.56,  Attention-deficit/hyperactivity disorder (ADHD) is characterized by a persistent pattern of inattention, impulsivity, and hyperactivity. It is one of the most prevalent neurodevelopmental disorders in children with prevalence rates varying from 5.9 to 7.1% . These sIn the recent decade, the prevalence rates of ADHD have shown a nearly 30% increase . As our Yet, although extensive research on the effects of maternal depression on offspring outcomes has started to emerge ,15, relaThese studies are, however, limited by a number of reasons. First, they measured depressive symptoms “during the past seven days or last two weeks” at one or two time points during pregnancy not covering the entire pregnancy ,20. SecoHence, we tested, in a large sample of pregnant Finnish women, if depressive symptoms, measured biweekly from gestational week 12 onwards until term or delivery, were associated with ADHD symptoms in their 3- to 6-year-old children. The biweekly assessments allowed us to address gestation-week and trimester-specific effects, and maternal re-ratings of depressive symptoms at the time of rating the 3- to 6-year-old child allowed us to address if any effects were specific to the prenatal stage. Our study also tested if maternal depressive symptoms after pregnancy added to or mediated any of the prenatal effects. Finally, we tested if maternal pre-pregnancy obesity, hypertensive pregnancy disorders, and gestational diabetes, or maternal ADHD symptoms accounted for any observed effects. We have previously demonstrated in this cohort associations between maternal depressive symptoms and child internalizing, externalizing, and total problems, including DSM IV-oriented ADHD problems  as measuThe Prediction and Prevention of Pre-eclampsia and Intrauterine Growth Restriction (PREDO) study comprises altogether 4,777 mothers and their singleton offspring born alive in Finland between 2006 and 2010 . The womSD) = 0.5 years, range 3.0 to 6.3 years; 51.5% boys).In 2011–2012 we invited 4,586 mother-child dyads , 55 women declined participation in a follow-up, and for 100 women, addresses were not traceable), and 2,667 (58.2%) participated. Of them 2,312 (68.0% of those with data on depressive symptoms during pregnancy) had pregnancy as well as follow-up data available at the child’s age of 1.9 to 6.3 years (50.6% boys). Since the CHI is validated for children who are 3 years and older , we excln = 2,274), the women who participated and whose children in the follow-up were 3 years and older  were older at delivery , had more often a tertiary education , were less often single , were less often multiparous , smoked less often throughout pregnancy , and reported less often a history of a depression diagnosis .Compared to the women who were invited but did not participate in the follow-up  to “very much” (3) . A sum-sDepressive symptoms were reported biweekly up to 14 times throughout pregnancy starting from 12+0–13+6 to 38+0–39+6 weeks+days gestation or delivery using the Center for Epidemiological Studies Depression Scale (CES-D) . The CESIn the follow-up, depressive symptoms were reported using the Beck Depression Inventory-II (BDI-II) . This scBoth depression scales have good psychometric properties –30, and 2), gestational diabetes (yes vs. no) and hypertensive pregnancy disorders  were extracted from the MBR and/or from medical records independently verified by a clinical jury.Maternal pre-pregnancy obesity  , which tWe first examined maternal depressive symptoms profiles during pregnancy with a latent profile analysis. We compared solutions with two to eight clusters, and identified the most optimal one by using Akaike Information Criterion, sample size-adjusted Bayesian Information Criterion, and Vuong-Lo-Mendell-Rubin Likelihood Ratio Test and Lo-Mendell-Rubin Adjusted Likelihood Ratio Tests. We then tested if the child ADHD symptom scores, treated as a continuous outcome variable, and the proportion of children with clinically significant ADHD symptoms, treated as a dichotomous variable using the ADHD symptom score 10 or above as a clinical cutoff , differeWe also examined if the associations between maternal depressive symptoms during pregnancy and child ADHD symptoms were gestation-week- or trimester-specific. In these tests, we used linear regression analysis when we treated child ADHD symptoms as continuous and logistic regression analysis when we dichotomized child ADHD symptoms scores at the clinical cutoff. Further, in these analyses maternal depressive symptom scores  were square root transformed to improve linear model fitting.In all of the above analyses we first made adjustments for child’s sex and age at follow-up (model 1). Thereafter, we additionally adjusted for maternal age at childbirth, parity, family structure, education level, type 1 diabetes, chronic hypertension, history of physician-diagnosed depression, antidepressant and other psychotropic medication use, alcohol use and smoking during pregnancy, and gestation length and weight at birth adjusted for sex and gestation length (model 2); for maternal pre-pregnancy obesity, gestational diabetes, gestational hypertension, and pre-eclampsia (model 3); for maternal ADHD problems (model4); and finally, for all of the above and maternal depressive symptoms at follow-up parallel to rating the child (model 5).We also tested if maternal depressive symptoms after pregnancy added to the prenatal effects with an interaction term of maternal trimester-weighted mean depressive symptoms during pregnancy*maternal depressive symptoms after pregnancy that was added to the linear (continuous ADHD symptom scores) and logistic regression models . In addition, we tested if maternal depressive symptoms after pregnancy mediated the effects of maternal trimester-weighted mean depressive symptoms during pregnancy using the PROCESS macro for mediation in SPSS 24 with 5000 bootstrapping re-samples with bias-corrected CIs ,33. Finap-values < 0.001) [Characteristics of the study participants are in < 0.001) . The medp-values < 0.001). For the two latent profile groups, the percentage of women with data on all 14 measurement points compared to the ones with at least one missing value was not significantly different . Further, higher maternal biweekly, trimester-specific  and trimp-values for interactions = 0.03 for depressive symptoms during pregnancy*depressive symptoms after pregnancy interaction on child continuous and clinically significant ADHD symptoms scores). Across all adjustment models, child ADHD symptom scores and proportion of children with clinically significant symptoms were the highest if the mother reported depressive symptoms above the clinical cutoff both during and after pregnancy  and 2.8-times higher odds for clinically significant ADHD symptoms. It was therefore not surprising that we found no gestation-week or trimester-specific associations between maternal depressive symptoms during pregnancy and child ADHD symptoms. None of these associations were accounted for by a number of perinatal, maternal and neonatal characteristics, and a series of sensitivity analyses demonstrated that the associations did not either vary by maternal pre-pregnancy obesity, hypertensive pregnancy disorders, or gestational diabetes, child’s sex, maternal history of physician-diagnosed depression, or maternal ADHD problems.Our study also showed that higher levels of maternal depressive symptoms after pregnancy were associated with higher child ADHD symptom scores. These higher levels of depressive symptoms after pregnancy only partially accounted for the prenatal effects as maternal depressive symptoms during pregnancy also had a significant direct effect on the child’s ADHD symptoms when adjusting for the symptoms after pregnancy. They did, however, add to the prenatal effects, such that child ADHD symptom scores and the proportion and odds of children with clinically significant ADHD symptoms were the highest among those women with clinically significant depressive symptoms both during and after pregnancy. Together maternal depressive symptoms during and after pregnancy accounted for 11% of the variation in the child’s ADHD symptoms.Our findings correspond with the Developmental Origins of Health and Disease (DOHaD) framework suggesting that prenatal exposure to environmental adversity may carry enduring effects on brain developmental sequelae, including risk for ADHD symptomatology ,13,34. OAn obvious study limitation is that we are not able to specify the brain structural or functional nor biological or behavioural underlying mechanisms. Existing literature suggests that higher maternal depressive symptoms and/or salivary cortisol levels during pregnancy are linked with altered offspring brain structure and functional connectivity , and witFurther study limitations relate to child ADHD symptoms being reported by the mother only. However, Leis et al. (2014) found that the effect of maternal prenatal depression on child hyperactivity was significant whether the child was rated by the mother or the teacher. Furthermore, we measured ADHD symptoms dimensionally, and did not use diagnostic criteria, rendering generalizations to ADHD disorder tentative. Since maternal depressive symptoms after pregnancy were self-rated at the time of rating the child’s behaviour, and maternal depression and child behaviour may influence each other, we cannot rule out shared method variance. Sample attrition which was not independent of maternal characteristics also limits the external validity of our findings.Our findings show that maternal depressive symptoms during and after pregnancy are associated with child ADHD symptomatology and suggest that early pregnancy screening and preventive interventions focusing on maternal depressive symptoms may benefit not only maternal, but offspring wellbeing. Preventive interventions, suited for pregnancy, are urgently needed, as a recent meta-analysis demonstrated null to very small benefits of existing techniques in decreasing maternal distress during pregnancy .S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.