The ongoing ascent of sequencing technologies has enabled researchers to gain unprecedented insights into the RNA content of biological samples. MiRNAs, a class of small non-coding RNAs, play a pivotal role in regulating gene expression. The discovery that miRNAs are stably present in circulation has spiked interest in their potential use as minimally-invasive biomarkers. However, sequencing of blood-derived samples  is challenging due to the often low RNA concentration, poor RNA quality and the presence of highly abundant RNAs that dominate sequencing libraries. In murine serum for example, the high abundance of tRNA-derived small RNAs called 5′ tRNA halves hampers the detection of other small RNAs, like miRNAs. We therefore evaluated two complementary approaches for targeted depletion of 5′ tRNA halves in murine serum samples. Using a protocol based on biotinylated DNA probes and streptavidin coated magnetic beads we were able to selectively deplete 95% of the targeted 5′ tRNA half molecules. This allowed an unbiased enrichment of the miRNA fraction resulting in a 6-fold increase of mapped miRNA reads and 60% more unique miRNAs detected. Moreover, when comparing miRNA levels in tumor-carrying versus tumor-free mice, we observed a three-fold increase in differentially expressed miRNAs. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression and play important roles in essential physiological and pathological processes3Several measurement technologies are available for assessing relative miRNA abundance: microarray, RT-qPCR and massively parallel sequencing. With differences in reproducibility, accuracy, sensitivity and specificity, the method of choice is strongly dependent on the specific research question789In serum, certain tRNA-derived fragments called 5′ tRNA halves appear to be abundantly present, consuming the majority of sequencing reads1112131516171819In this study, we evaluated two complementary approaches for selective depletion of 5′ tRNA halves from murine serum derived RNA based on bead capturing and RNase H cleavage, respectively. Using bead-based depletion, we were able to reduce the 5′ tRNA halves isotypes by more than 95%. This enabled us to increase the number of reads mapping to miRNAs by 6-fold, with 60% more unique miRNAs detected and importantly, with no differential effects on the constitution of the remaining miRNA population. To demonstrate the benefit of performing a 5′ tRNA half depletion, we investigated differentially expressed miRNAs between tumor-free mice and mice carrying orthotopic xenografts. In line with a higher detection rate, the selective depletion of 5′ tRNA halves increased the detectable amount of differentially expressed miRNAs by a factor of three compared to the same non-depleted RNA samples.First, we evaluated the effect of the small RNA library preparation method on the 5′ tRNA-halves abundance in libraries prepared from serum RNA using three commercially available small RNA library preparation kits. RNA isolated from 100 μl of serum collected from healthy mice was used as input for the preparation of a small RNA library in duplicate and libraries were single end sequenced.Even though all libraries were sequenced at the same sequencing depth, we observed a clear difference in the number of mapped reads between the different library prep kits. Using the NEBNext kit we obtained between 17.1 and 26.4 million mapped reads, with the TruSeq kit between 10.0 and 12.5 million mapped reads and with the TailorMix kit between 5.3 and 5.5 million mapped reads. Annotation analysis of the mapped sequencing reads revealed that all libraries contained high amounts of reads mapping to tRNA genes, irrespective of library preparation method . We founInvestigation of the different types of 5′ tRNA halves present in the libraries revealed that the majority of 5′ tRNA half reads corresponded to just a small subset of tRNAs types. The most abundantly present tRNA types are those with glutamine, glycine, valine and histidine isoacceptors. The relative percentages of these tRNA types vary between the library preparation kits, but their total relative contribution is similar and close to 95% of all tRNA reads .Next, we evaluated two complementary protocols for 5′ tRNA half depletion for the four tRNA types that we found to be most dominantly present in murine serum: glutamine, glycine, valine and histidine.For each tRNA half type, different isoacceptors were detected. Comparison of the sequences of the different isotypes showed that it was possible to effectively deplete these fragments using only a limited number of probes for each tRNA type. We designed DNA probes complementary to the most abundantly detected sequences of the four most abundant tRNA half types. In total we designed five probes for depletion of the four most abundant tRNA halves. To interrogate for possible off-target effects, probe sequences were queried against the human and mouse miRNome to identify miRNAs that show perfect complementarity with the probes . In totaTwo complementary approaches were evaluated for their efficacy in depleting 5′ tRNA-halves . In a fiBoth protocols were evaluated on RNA isolated from a serum pool that was collected from healthy SVj mice by cardiac puncture. We assessed the 5′ tRNA half depletion efficiency using RT-qPCR and compared tRNA-half levels between depleted and non-depleted samples . For botTo assess whether the depletion protocols could affect miRNA expression values, we determined the abundance levels of mmu-miR-16-5p , a miRNA that is reported to be highly and stably expressed in mouse serum, in depleted and control samplesTo evaluate the efficacy of the described protocols on small RNA sequencing, we performed the two 5′ tRNA halves depletion protocols on total RNA isolated from a serum pool collected from healthy mice. Each RNA sample was divided into three and the resulting fractions were either depleted using beads or RNase H, or used as a control. The experiment was performed in triplicate. Upon depletion, libraries were prepared using TruSeq Small RNA library preparation kit (given the higher number of mapped reads and miRNA reads as compared to the NEBNext and TailorMix kits). Samples were sequenced as described above.6, 1.77 × 105 and 5.46 × 106 for control, RNase H-depleted and beads-depleted samples, respectively. Comparison of the relative abundance of the different RNA species revealed a pronounced reduction in the fraction of detected 5′ tRNA halves in depleted samples as compared to non-depleted samples  in depleted samples . Of the Low RNA concentration due to its low abundance in serum poses a critical obstacle for the exact quantification of serum-derived miRNAs, especially for sequencing-based technologies. There have been several efforts to improve discrete steps of the miRNA quantification process in order to enhance the efficacy of miRNA detection in these types of samples. As such, improvement of pre-analytical parameters such as storage conditions and serum preparation, optimization of the RNA extraction process and advancements in library preparation protocols were found to improve miRNA detection performance25262728In this study we optimized and compared two protocols for the selective depletion of 5′ tRNA halves in RNA isolated from murine serum samples of which only one protocol was successful in targeted depletion of 5′ tRNAs halves. Using a protocol consisting of target capture by biotinylated DNA probes followed by immobilization using magnetic streptavidin beads, we were able to successfully deplete up to 99% of the targeted 5′ tRNA halves in RNA samples. Depletion of 5′ tRNA halves in small RNA libraries resulted in a more than 6-fold increase in mapped miRNA reads and 60% more detected mature miRNAs when performing small RNA sequencing on murine serum samples. Importantly, we observed no considerable effects on the relative expression values of miRNAs. Moreover, using 5′ tRNA halves depletion we were able to detect 3 times more differentially expressed miRNAs when comparing serum from mice carrying orthotopically engrafted tumors with serum from healthy control mice. We detected 34 differentially expressed miRNAs of which six are unique to humans, 13 unique to mice and 15 have complete conservation between the two species. Especially for such applications, in which the miRNA sequence itself can carry information about its cellular origin, sequencing is very well suited because of its exquisite specificity when strict mapping is applied. In contrast, RNase H-based depletion did not prove highly effective. It could be that cleaved tRNA half fragments are still being processed during library preparation as RNAse H can yield polished, easily ligated RNA ends.It is straightforward to further increase the efficacy of 5′ tRNA halves depletion by including additional capture probes that target other 5′ tRNA isoacceptors not targeted in this study. It has been reported that the expression of circulating 5′ tRNA halves derived from specific tRNA isoacceptors can change as a result of experimental conditions like aging and calorie restrictionHealthy male and female 129/SVj mice, with ages varying between 2 to 9 months, were sedated using tribromoethanol at a dose of 250 mg/kg injected intraperitoneally. 500 μl to 1 ml of blood per mouse was collected in Safe-Lock micro test tubes (Eppendorf) by cardiac puncture using a 0.45 × 12 mm needle after which mice were sacrificed by cervical dislocation. For NCr nude mice (Taconic Farms), 100 μl blood was collected by puncture of the jugular vein using a 4 mm lancet and collected in a BD Vacutainer collection tube with a gel separator (BD). All blood samples clotted at room temperature for 45 min. Clotted blood samples from healthy SVj mice were centrifuged at 1900 g for 10 min at 4 °C using a swinging bucket rotor. The supernatant, containing the serum, was collected and centrifuged again at 16000 g for 10 min at 4 °C in a fixed angle rotor to pellet cellular nucleic acids attached to cell debris. The supernatant was collected and stored at −80 °C. Clotted blood samples of nude mice were centrifuged at 2000 g for 15 minutes at 4 °C in a fixed angle rotor. The supernatant was collected and stored at −80 °C. For all serum samples, the degree of hemolysis was determined by measuring levels of free hemoglobin by spectral analysis. Absorbance peaks at 414, 541 and 576 nm are indicative of free hemoglobin6 human SH-SY5Y neuroblastoma cells were surgically implanted in beneath the renal capsule. This model closely resembles the growth characteristics of primary neuroblastoma arising from the para-adrenal location in humansOrthotopic neuroblastoma xenografts were generated in four to six week old female athymic immunodeficient NCr nude mice as previously describedRNA was isolated using the miRNeasy serum/plasma kit (Qiagen) according to the manufacturer’s instructions. For the final experiment, 50, 100 or 200 μl of serum was used as input and total RNA was eluted in 12 μl of RNAse-free water.RNA used for the evaluation of the different library preparation kits was isolated from 100 μl of serum collected from individual SVj mice and eluted in 12 μl of RNAse-free water. For RNA isolation of samples used for the assessment of effective tRNA depletion by sequencing, serum samples from 3 individual SVj mice were pooled and 200 μl aliquots of the serum pool were used as input for RNA isolation and total RNA was eluted in 12 μl of RNAse-free water. After elution from the miRNeasy column, RNA samples used for the assessment of tRNA depletion by sequencing were diluted to a final volume of 38 μl and distributed in 12 μl aliquots. RNA used for differential expression analysis was isolated from 50 μl of serum that was collected from Ncr nude mice and eluted in 12 μl of RNAse-free water.5′ biotinylated DNA probes were designed for four 5′ tRNA halves species  and ordeFor tRNA halves depletion using beads, 12 μl of RNA, 15 μl of 2x hybridization buffer , 1 μl ofFor tRNA halves depletion using RNAse H, 2 μl of 7.5x hybridization buffer  was combFor reverse transcription we used the miScript II RT kit (Qiagen) with HiFlex buffer according to manufacturer’s instructions but with 1.5 μl of input RNA. After reverse transcription, the resulting cDNA was diluted 22-fold by adding 210 μl nuclease-free water to 10 μl of RT product.For detection of tRNA-halves by RT-qPCR the miScript SYBR green PCR kit (Qiagen) was used with custom designed primer assays . The RT-For Small RNA library preparation we used the TailorMix miRNA sample preparation kit v2 (Seqmatic), the NEBNext Multiplex Small RNA library prep kit (New England Biolabs) or the TruSeq small RNA library preparation kit v2 (Illumina) following manufacturer’s instructions with small modifications . AdaptorFor RT-qPCR quantification of sequencing libraries, 1 μl of EtOH purified library was diluted 1:100 000. 2.5 μl of SsoAdvanced universal SYBR green supermix (Bio-Rad Laboratories) and 0.25 μl of each primer 5 μM;  were comFor the quantification of small RNAs Biogazelle’s dedicated small RNA seq pipeline was used (part of Cobra), as publically available alternatives we advice the use of miRDeep2 and miRExpress. Adaptor trimming was performed using Cutadapt v1.8.1, reads shorter than 15 bp and those in which no adaptor was found, were discarded. For quality control the FASTX-Toolkit (v0.0.14) was used, a minimum quality score of 20 in at least 80% of bases was applied as a cutoff. The reads were mapped with Bowtie (v1.1.2) without allowing for mismatches. Mapped reads were annotated by matching genomic coordinates of each read with genomic locations of miRNAs  and other small RNAs  and Ensembl, v84). Raw and processed sequencing data have been submitted to the GEO database (GSE88915). Further data analysis was performed using RStudio . Normalization of miRNA counts and differential expression analysis was performed using the R package DESeq2 (v1.8.2). The DESeq2 result tables are available in How to cite this article: Van Goethem, A. et al. Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples. Sci. Rep.6, 37876; doi: 10.1038/srep37876 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We design and construct a three-dimensional (3D) negative index medium (NIM) composed of gold hemispherical shells to supplant an integration of a split-ring resonator and a discrete plasmonic wire for both negative permeability and permittivity at THz gap. With the proposed highly symmetric gold hemispherical shells, the negative index is preserved at multiple incident angles ranging from 0° to 85° for both TE and TM waves, which is further evidenced by negative phase flows in animated field distributions and outweighs conventional fishnet structures with operating frequency shifts when varying incident angles. Finally, the fabrication of the gold hemispherical shells is facilitated via standard UV lithographic and isotropic wet etching processes and characterized by μ-FTIR. The measurement results agree the simulated ones very well. The advent of negative index media (NIM)135181112To resolve such circumscription, in this work we demonstrate a simple 3D NIM composed of an array of monolithic metallic hemispherical shells, which allows both negative permeability and negative permittivity under multiple-angle incidences within the THz gap. Besides, the employment of the metallic hemispherical shell guarantees a polarization independent response due to its circular symmetry under normal-incidence excitation. Most importantly, different from the antecedent 3D NIM1416TMCST Microwave Studio, to calculate the complex scattering parameters of our proposed 3D NIM and then examine the retrieval results from the effective medium theoryTo verify our design strategy, we employ a finite-integration solver of As for the fabrication of the 3D NIM, one can follow procedures as illustrated in Finally, we utilized a micro Fourier transform infrared spectrometer (Bruker Vertex 70 V μ-FTIR) to measure the relative transmission of the fabricated samples at 0°, 15° and 30° incidences by our homemade stage. These samples were illuminated by a mercury lamp through an aperture of 8 mm in diameter, and a PE/DTGS detector was placed on the opposite side to collect the transmitted signals. All measurements were done in vacuum to reduce the noise from the ambient environment including absorption by moisture and air.Our proposed 3D NIM, the gold hemispherical shell is presented in 19TMCST Microwave Studio, to calculate the complex scattering parameters. For the metallic hemispherical shell, we chose an excellent conductor of gold with conductivity (σ) of 4.56×107 S/m due to its lower losses in the THz region and inert chemical properties beyond copper and silver, respectively; for the supporting substrate, we adopted a 50-μm-thick silicon (εr = 12 and σ = 8 S/m)1314To validate and to characterize our design, we employed a To certify our prediction, the corresponding constitutive parameters are retrieved to verify the existence of the double negative identities, i.e., a negative indexTo further interpret the magnetic and electric resonances in the monolithic gold hemispherical shell, we scrutinize the distributions of surface currents at the resonance frequency  as illustrated in Finally, we monitor the animated field distribution of the gold hemispherical shells to identify the negative index by revealing the opposite propagation direction of the phase flows across the NIM and free space24The success in developing the 3D NIM under the normal incident condition by the gold hemispherical shell array encourages us to further examine the shell under oblique incident conditions. In the following, we would elaborate transmission apexes of the single-layered 3D NIM to identify the double negative identities under oblique incidence from 0° up to 85° for both TE and TM cases see 26. On the 113Next, we record the phase flow of within the 3D NIM under the oblique incident cases and exemplify the negative index at 15° and 30° incident angles for both TE and TM cases. The transmission spectra and animated phases of the five-layered 3D NIM at 15° and 30° incidences are portrayed in Note that this 3D NIM possesses a negative index at least from 0° to 85° incidences for both TE and TM cases  with a homemade platform to control various incident angles. The measured transmittance spectrum of the gold hemispherical shell under normal incidence is shown in We demonstrated an NIM by the monolithic gold hemispherical shells in both numerical simulation and experimental verification. Such monolithic gold hemispherical shells are the three-dimensional superposition of conventional SRRs for negative permeability and discrete plasmonic wires for negative permittivity, allowing a negative index operated at multiple-angle incidences. Due to the circular symmetry of the hemispherical shell, this deliberately designed NIM is insensitive to the polarization of incident light; meanwhile, the 3D NIM maintains its negative index under the oblique incident angles spanning from 0° to 85° for both TE and TM cases, evidenced by the negative phase flows within the five-layered shells. Our designed monolithic gold hemispherical shells are fabricated via standard UV lithographic and isotropic wet etching processes, and then measured by an evacuated μ-FTIR system. Both simulation and experiment were in a good agreement. To our best knowledge, this is the very first work to experimentally demonstrate a feasible 3D NIM at THz ranges. Such an NIM comprised of monolithic gold hemispherical shells can ease the burden of the strong anisotropic responses in the conventional planar metamaterials and widens the route to practical applications including superlenses, slowing light by negative Goos-Hänchen effect and perfect absorbers.How to cite this article: Yeh, T.-T. et al. Demonstration of a Three-dimensional Negative Index Medium Operated at Multiple-angle Incidences by Monolithic Metallic Hemispherical Shells. Sci. Rep.7, 45549; doi: 10.1038/srep45549 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Schizophrenia is a highly heritable, neuropsychiatric disorder characterized by episodic psychosis and altered cognitive function. Despite success in identifying genetic variants associated with schizophrenia, there remains uncertainty about the causal genes involved in disease pathogenesis and how their function is regulated.We performed a multi-stage epigenome-wide association study, quantifying genome-wide patterns of DNA methylation in a total of 1714 individuals from three independent sample cohorts. We have identified multiple differentially methylated positions and regions consistently associated with schizophrenia across the three cohorts; these effects are independent of important confounders such as smoking. We also show that epigenetic variation at multiple loci across the genome contributes to the polygenic nature of schizophrenia. Finally, we show how DNA methylation quantitative trait loci in combination with Bayesian co-localization analyses can be used to annotate extended genomic regions nominated by studies of schizophrenia, and to identify potential regulatory variation causally involved in disease.This study represents the first systematic integrated analysis of genetic and epigenetic variation in schizophrenia, introducing a methodological approach that can be used to inform epigenome-wide association study analyses of other complex traits and diseases. We demonstrate the utility of using a polygenic risk score to identify molecular variation associated with etiological variation, and of using DNA methylation quantitative trait loci to refine the functional and regulatory variation associated with schizophrenia risk variants. Finally, we present strong evidence for the co-localization of genetic associations for schizophrenia and differential DNA methylation.The online version of this article (doi:10.1186/s13059-016-1041-x) contains supplementary material, which is available to authorized users. P < 5 × 10−8), with evidence for a substantial polygenic component in signals that fall below this stringent level of significance . We identified a significant enrichment  in certain specific transcription factor binding motifs including BATF , BCL11A , IRF4 (M-17) , and MEF2A , and a significant depletion in HA-E2F1 binding-sites . In order to investigate functional relationships between the 955 genes annotated to the 1223 phase 1 discovery DMPs, we tested for an over-representation of ontological categories and pathways using a method that controls for the number of probes annotated to each gene on the 450 K array  DMPs identified in phase 1 were characterized by highly consistent schizophrenia-associated differences in the same direction in the phase 2 dataset (sign test P = 7.75 × 10−7)  differences in the same direction in phase 2, with five additional DMPs significant at P < 5 × 10−5. The phase 1 discovery DMPs (P < 5 × 10−5) were also characterized by highly consistent schizophrenia-associated differences in phase 2, with 1159 (94.8 %) having a consistent direction of effect (sign test P = 4.25 × 10−261). Of the phase 1 DMPs, 137 (11.2 %), 249 (20.4 %), and 245 (20.0 %) were associated at P < 1 × 10−7, P < 5 × 10−5, and P < 4.09 × 10−5 (correcting for 1223 DMPs) respectively  by generating 450 K array data from an additional 414 patients with schizophrenia and 433 non-psychiatric controls. As with the phase 1 cohort, patients with schizophrenia were characterized by a significantly higher smoking score derived from Illumina 450 K array DNA methylation data (Mann–Whitney −7) Fig. ; 14 of t by generP < 1 × 10−7) reached experiment-wide significance in the twin dataset  and 1113 out of 1216 (91.5 %) phase 1 discovery DMPs  were characterized by schizophrenia-associated differences in the same direction, demonstrating that these effects were not confounded by factors such as genotype and sex that are perfectly matched between genetically identical twins. Finally, a meta-analysis across the three independent datasets demonstrated that 22 out of 25 (88 %) of the phase 1 experiment-wide significant DMPs were characterized by an experiment-wide significant association across all cohorts, with an additional 343 experiment-wide DMPs (P < 1 × 10−7) identified in the combined meta-analysis ; the analysis of MZ twins is a powerful tool in epigenetic epidemiology, as they do not differ for many of the confounders that can influence case–control analyses  [; the anaP = 3.34 × 10−27; Additional file r = 0.963–0.980) with the effects identified in the initial EWAS , with 156 DMPs identified at the more relaxed discovery threshold of P < 5 × 10−5 , in which patients with schizophrenia were again characterized by a significantly higher schizophrenia PRS than controls  that demonstrated significant differences in DNA methylation . Briefly, this approach compares the pattern of association results from two independent GWAS (i.e. of schizophrenia and DNA methylation) to see if they are indexing an association with the same causal variant. We considered mQTL data for 23,649 unique Illumina 450 K probes located within 500 kb of the 105 autosomal GWAS-nominated regions defined by the PGC [3 + PP4 > 0.99; Additional file 4/PP3 > 1), with 66 (over 27 regions) of these having sufficient support for them to be considered as “convincing” (PP4/PP3 > 5) according to the criteria of Guo and colleagues [3 + PP4 > 0.99) for both schizophrenia and DNA methylation in blood, seven (44 %) of which were also classed as demonstrating convincing co-localization with blood mQTLs  from European twin studies based in Utrecht (The Netherlands), Helsinki (Finland), London (UK), Stockholm (Sweden), and Jena (Germany). Of the MZ twin pairs utilized in the analysis, 75 were discordant for diagnosed schizophrenia, six were concordant for schizophrenia, and 15 twin pairs were free of any psychiatric disease. In this analysis we tested specific DNA methylation probes nominated from our case–control analysis; a more detailed description of the cohort along with more in-depth analysis is currently under preparation .The MZ twin cohort is a multi-center collaborative project aimed at identifying DNA methylation differences in MZ twin pairs discordant for schizophrenia. We identified 96 informative twin-pairs  was used to treat 500 ng of DNA from each sample with sodium bisulfite in duplicate. DNA methylation was quantified using the Illumina Infinium HumanMethylation450 BeadChip (Illumina Inc.) run on an Illumina iScan System (Illumina) using the manufacturers’ standard protocol. Samples were randomly assigned to chips and plates to ensure equal distribution of cases and controls across arrays and to minimize batch effects. In addition, a fully methylated control  was included in a random position on each plate.methylumIDAT function in the methylumi package [https://dnamage.genetics.ucla.edu/) [pfilter function from wateRmelon package [P value > 0.05 and probes with >1 % of samples with detection P value > 0.05. PCs were used  to identify outliers, samples >2 standard deviations from the mean for both PC1 and PC2 were removed. Finally, we checked the correlation (r = 0.927) of reported age with that predicted by the Epigenetic Clock. Normalization of the DNA methylation data was performed used the dasen function in the wateRmelon package [Signal intensities were imported in the R programming environment using the  package . Our strla.edu/) ; (5) mul package  to exclu package . Due to Genotyping was performed using the Affymetrix Mapping 500 K Array and the Genomewide Human SNP Array 5.0 or 6.0 . Genotypes were called from raw intensity data using the Birdseed component of the Birdsuite algorithm , 54. SamP value < 0.001 and a minor allele frequency of <5 %. Imputation was performed using ChunkChromosome (http://genome.sph.umich.edu/wiki/ChunkChromosome) and Minimac2 [P-value < 0.001, and a minor allele frequency of >5 %. Subsequently, SNPs were also filtered so that each of the three genotype groups with zero, one, or two minor alleles  had a minimum of five observations.Prior to imputation, PLINK  was usedMinimac2 , 57 withMinimac2  only incAs smoking status information was not present for all samples, we estimated a proxy based on the DNA methylation profile at sites known to be associated with smoking status following the approach in . This meP value < 0.05.As samples from both phase 1 and phase 2 were included in the PGC GWAS of schizophrenia, we obtained the PRS scores from these analyses calculated as part of the leave one out validation experiment, where the training dataset (to derive weights for associated scores) was based on all samples bar one source dataset in which the PRSs were calculated [P-values. DMP results are annotated with their genomic location and gene annotation taken from the annotation files provided by Illumina. In addition, transcription factor binding site and DHS site annotation were taken from the supplementary files provided by Slieker and colleagues [Probes previously identified as containing a common SNP  within 10 base pairs (bp) of the single base extension position  or potenlleagues .P values from the EWAS were saved and the minimum identified. Across all permutations the fifth percentile was calculated to generate the 5 % alpha significance threshold.To establish the multiple testing significance threshold, 5000 permutations were performed repeating the linear regression model for randomly selected groups of cases and controls to match the numbers in the phase 1 data. For each permutation, P value) and run through the comb-p [−5 and distance parameter set to 500 bp. Briefly, comb-p generates DMRs by (1) calculating the auto-correlation between probes to adjust the input DMP P-values using the Stouffer–Liptak–Kechris correction, (2) running a peak finding algorithm over these adjusted P values to identify enriched regions around a seed signal, (3) calculating the region P value using the Stouffer–Liptak correction, and (4) correcting for multiple testing with the one-step Šidák correction. Significant regions were identified as those with at least two probes and a corrected P value < 0.05.Two different region approaches were used. First, the results for every probe were converted into a BED file , before moving to the next most significant region, until the bottom of the list was reached.Second, we implemented a sliding window approach with multiple window sizes. Previous EWAS have reported DMRs that span either a few hundred or a few thousand base pairs . As is in probes . Each pratistics . To deriPublished 450 K array probe annotations  were usehttp://geneontology.org/) and mapped to genes, including all parent ontology terms. All genes with at least one 450 K probe annotated and mapped to at least one GO pathway were considered. Pathways were filtered to those containing between 10 and 2000 genes. After applying this method to all pathways, the list of significant pathways (P < 0.05) was refined by grouping to control for the effect of overlapping genes. This was achieved by taking the most significant pathway, and retesting all remaining significant pathways while controlling additionally for the best term. If the test genes no longer predicted the pathway, the term was said to be explained by the more significant pathway, and hence these pathways were grouped together. This algorithm was repeated, taking the next most significant term, until all pathways were considered as the most significant or found to be explained by a more significant term.Illumina UCSC gene annotation, which is derived from the genomic overlap of probes with RefSeq genes or up to 1500 bp of the transcription start site of a gene, was used to create a test gene list from the DMPs for pathway analysis. Where probes were not annotated to any gene (i.e. in the case of intergenic locations), they were omitted from this analysis; where probes were annotated to multiple genes, all were included. A logistic regression approach was used to test if genes in this list predicted pathway membership, while controlling for the number of probes that passed quality control  annotated to each gene. Pathways were downloaded from the GO website . Briefly, these were identified by performing a “clumping” procedure on the GWAS P values to collapse multiple correlated signals (due to LD) surrounding the index SNP  into a single associated region. To define 108 physically distinct loci, those within 250 kb of each other were subsequently merged to obtain the final set of GWAS regions. The outermost SNPs of each associated region define the start and stop parameters of the region. Using the set of 105 autosomal schizophrenia-associated genomic loci we used Brown’s method [P value across all 450 K probes located within each region. This used the P values from both the case–control and PRS EWAS and correlation coefficients between all pairs of probes calculated from the DNA methylation values. This methodology was repeated with the 5000 random permutations we generated. Empirical P values for each region were calculated by counting how many of the permutations had more significant P values than the true combined P value and dividing by the total number of permutations performed.The GWAS regions were defined by the PGC in their original manuscript  and are s method  to calcucis mQTLs , all DNA methylation sites located within 500 kb of these regions were identified and cis mQTL analysis was performed using MatrixEQTL [Schizophrenia-associated genomic loci were taken as the 105 autosomal regions published as part of the PGC mega-analysis . Given otrixEQTL . An addihttp://cran.r-project.org/web/packages/coloc) for each DNA methylation site within each region. From both the PGC schizophrenia GWAS data and our mQTL results we inputted the regression coefficients, their variances, and the SNP minor allele frequencies, and the prior probabilities were left as their default values. This methodology quantifies the support across the results of each GWAS for five hypotheses by calculating the posterior probabilities, denoted as PPi for hypothesis Hi:H0: there exist no causal variants for either trait;H1: there exists a causal variant for one trait only, schizophrenia;H2: there exists a causal variant for one trait only, DNA methylation;H3: there exist two distinct causal variants, one for each trait;H4: there exists a single causal variant common to both traits.Co-localization analysis was performed as previously described  using th450K array, Illumina Infinium HumanMethylation450 BeadChip; bp, Base pair; DHS, DNAse I hypersensitivity site; DMP, Differentially methylated positions; DMR, Differentially methylated region; DSM-IV, Diagnostic and Statistical Manual for Mental Disorders fourth edition; EWAS, Epigenome-wide association study; GO, Gene Ontology; GWAS, Genome-wide association study; ICD-10, International Classification of Diseases 10th edition; LD, linkage disequilibrium; MHC, Major histocompatibility complex; mQTL, DNA methylation quantitative trait loci; MZ, Monozygotic; OPCRIT, Operational Criteria Checklist; OR, Odds ratio; PC, Principal component; PGC, Psychiatric Genomics Consortium; PRS, Polygenic risk score; RDC, Research diagnostic criteria; SADS-L, Schedule for Affective Disorders and Schizophrenia-Lifetime Version; SCID, Structured Clinical Interview for DSM-IV; SNP, single nucleotide polymorphism


Originating from the investigation of condensed matter states, the concept of quantum Hall effect and quantum spin Hall effect (QSHE) has recently been expanded to other field of physics and engineering, e.g., photonics and phononics, giving rise to strikingly unconventional edge modes immune to scattering. Here, we present the plasmonic analog of QSHE in graphene plasmonic crystal (GPC) in mid-infrared frequencies. The band inversion occurs when deforming the honeycomb lattice GPCs, which further leads to the topological band gaps and pseudospin features of the edge states. By overlapping the band gaps with different topologies, we numerically simulated the pseudospin-dependent one-way propagation of edge states. The designed GPC may find potential applications in the fields of topological plasmonics and trigger the exploration of the technique of the pseudospin multiplexing in high-density nanophotonic integrated circuits. Photonic topological insulators –4, opticSurface plasmon polaritons (SPPs) , elementIn this work, we theoretically explore the topological properties of two-dimensional (2D) graphene plasmonic crystals (GPCs) constructed by periodically arranged graphene nanodisks. Dirac cones at the Brillouin zone (BZ) corners are folded to a double Dirac cone at the BZ center by utilizing the zone folding mechanism. In order to obtain topological band gaps, we take further deformations on honeycomb lattice. By shrinking or expanding the graphene nanodisks, the double Dirac cone is opened and the band inversion occurs between pseudospin dipole and quadrupole modes, which further leads to topological phase transition between nontrivial and trivial states. Furthermore, one-way propagation of edge states is numerically simulated along an interface constructed by the trivial and nontrivial GPCs, which further demonstrates the pseudospin characteristics and topological robustness of our designed plasmonic crystals.a = 40 nm, μc1, and r are the chemical potential and radii of the graphene nanodisks; μc2 denotes the chemical potential of the surrounding graphene. By solving the Maxwells equations with boundary conditions, we obtain the dispersion relation for transverse magnetic (TM)-polarized SPP modes supported on the graphene layer surrounding by air and silica  can be expressed as [k± = kx ± iky, and A comes from off-diagonal elements of the first-order perturbation term α = 1, 2 and β = 3, 4. The effective Hamiltonian Heff(k) takes a similar form as the Bernevig-Hughes-Zhang (BHZ) model for the CdTe/HgTe/CdTe quantum well system [To further explore the topological property of the band gaps shown in Fig. essed as , 35.5\dol system , implyinl system .6\documeM = (Ep – Ed)/2 is the frequency difference between E2 and E1 representations at the Γ point. B is determined by the diagonal elements of the second-order perturbation term and is typically negative [C± = 0 is obtained when having a normal band order as displayed in Fig. M becomes positive when band inversion occurs. Therefore, C± = ±1 is simply obtained, and the gap in Fig. Here, negative . Thus, CΓK direction for such a ribbon, where a bulk band gap is spanned by additional topological edge states as indicated by the double degenerated red curves. Fig. kx = − 0.05π/a) and B (with kx = 0.05π/a) marked in Fig. By overlapping the band gaps with different topologies , one can create edge states that are spatially confined around the interface between two plasmonic crystals. Here, we consider a ribbon of topologically nontrivial plasmonic crystal  of anticlockwise (clockwise) circular polarization of in-plane magnetic field. One of the most distinguishing features of topological edge states is that they are robust against perturbations/imperfections. To verify this robustness, we construct sharp bends as displayed in Fig. S−. The SPP wave vanished eventually after a long traveling distance along the sharp bends due to the intrinsic loss of graphene material. To further confirm this topological transmission, we also exhibit the electric field intensity distribution by ignoring the intrinsic loss of graphene for comparison. As can be seen from Fig. The pseudospin-dependent unidirectional transmission of edge states are also demonstrated in a finite 20In summary, we have systematically investigated the band topologies of the GPCs constructed by periodically patterned graphene nanodisks. By employing zone folding mechanism, the Dirac cones at the BZ corner are folded to a double Dirac cone at the BZ center. Further, topological band gaps are realized by deforming the honeycomb lattice GPCs. Based on the effective Hamiltonian derived by the


Plasmodium vivax is a significant contributor to the global malaria burden, and a vaccine targeting vivax malaria is urgently needed. An understanding of the targets of functional immune responses during the course of natural infection will aid in the development of a vaccine. Antibodies play a key role in this process, with responses against particular epitopes leading to immune selection pressure on these epitopes. A number of techniques exist to estimate levels of immune selection pressure on particular epitopes, with a sliding window analysis often used to determine particular regions likely to be under immune pressure. However, such analysis neglects protein three-dimensional structural information. With this in mind, a newly developed tool, BioStructMap, was applied to two key antigens from Plasmodium vivax: PvAMA1 and PvDBP Region II. This tool incorporates structural information into tests of selection pressure.PvAMA1 and PvDBP. Structural patterns of nucleotide diversity were similar across all populations examined, with Domain I of PvAMA1 having the highest nucleotide diversity and displaying significant signatures of immune selection pressure (Tajima’s D > 0). Nucleotide diversity for PvDBP was highest bordering the dimerization and DARC-binding interface, although there was less evidence of immune selection pressure on PvDBP compared with PvAMA1. This study supports previous work that has identified Domain I as the main target of immune-mediated selection pressure for PvAMA1, and also supports studies that have identified functional epitopes within PvDBP Region II.Sequences from a number of populations were analysed, examining spatially-derived nucleotide diversity and Tajima’s D over protein structures for P. vivax, to examine structural patterns of selection and diversity across a number of geographic populations. There were striking similarities in structural patterns of diversity across multiple populations. Furthermore, whilst regions of high diversity tended to surround conserved binding interfaces, a number of protein regions with very low diversity were also identified, and these may be useful targets for further vaccine development, given previous evidence of functional antibody responses against these regions.The BioStructMap tool was applied to leading vaccine candidates from The online version of this article (10.1186/s12936-018-2324-3) contains supplementary material, which is available to authorized users. Plasmodium vivax infected an estimated 8.55 million people in 2016 and is a significant contributor to global malaria morbidity, with the majority of P. vivax cases occurring within South-East Asia [P. vivax, and an understanding of the targets of natural immune responses following P. vivax infection is likely to aid such an effort. A key challenge in vaccine development is the identification of specific antigens and epitopes that are targets of protective antibody responses. It is possible to use population genetic data to identify regions of proteins that are under immune-mediated selection pressure, which gives rise to balancing selection within that protein region. Tajima’s D is one test statistic that is often used to identify departures from a neutral model of selection, and has been applied to malaria genes both on a per-gene basis [P. vivax proteins such as apical membrane antigen 1 (PvAMA1) and Duffy-binding protein (PvDBP) for evidence of immune selection pressure using these approaches [P. vivax vaccine candidates: PvAMA1 and PvDBP.ne basis , or as ane basis –7. A numproaches , 8–11. Hproaches , and hasPlasmodium species [Plasmodium AMA1 proteins is divided into three domains, termed Domains I (DI), II (DII) and III (DIII)  on human reticulocytes during invasion [PvDBP is not absolutely essential for invasion of reticulocytes [P. vivax infection, and hence PvDBP makes an attractive vaccine target [PvDBP is part of the erythrocyte-binding like (EBL) family of proteins, which include EBA175, EBA181 and EBA140 in P. falciparum [PvDBP is the sole EBL family protein in P. vivax [PvDBP binds to DARC via its DBL domain (RII) [PvDBP molecules form a dimer around two DARC molecules [PvDBP has been divided into a number of subdomains (subdomains 1–3) [PvDBP have been associated with protection from clinical malaria in naturally exposed cohorts [PvDBP RII epitopes have been found to inhibit both attachment of PvDBP RII to erythrocytes [PvDBP, a study of 100 Sri Lankan isolates found no evidence of significant selection pressure on this region using Tajima’s D, dN/dS or Fu and Li’s D and F statistics [PvDBP RII across multiple populations showed a significantly positive value of dN/dS in this region, suggesting that this region may be under immune selection pressure [invasion , 29. Whiulocytes –32, Duffe target . PvDBP ilciparum . PvDBP iP. vivax . EBL famP. vivax –38; PvDBin (RII) . During olecules , 40. RIIins 1–3)  (Additioins 1–3) . Immune  cohorts , 43 whilhrocytes  and in vhrocytes . With reatistics . Anotherpressure .PvAMA1 and PvDBP Region II were examined in the context of protein structure, using a newly developed tool called BioStructMap [PfAMA1 bordering DII and DIII that had a strong signature of balancing selection when considering spatially derived Tajima’s D [PvAMA1, rather than DII or DIII, as being under balancing selection, it was considered that incorporation of protein structural information might yield additional insights into other regions under immune selection pressure. Genomic sequences from a number of populations were analysed, and spatially-derived nucleotide diversity and Tajima’s D were examined using protein structural information for PvAMA1 and PvDBP Region II. Structural patterns of nucleotide diversity were similar across all populations examined, with Domain I of PvAMA1 having the highest nucleotide diversity and displaying significant signatures of balancing selection (Tajima’s D > 0). Nucleotide diversity for PvDBP was highest bordering the dimerization and DARC-binding interface, although there was less evidence of immune selection pressure on this antigen.In this study, selection pressures on tructMap . BioStrujima’s D . Given tPvDBP (PVX_110810) and PvAMA1 (PVX_092275) were obtained from PlasmoDB, v34 (http://www.plasmoDB.org) [P. vivax, and with the condition that sequences had to cover > 95% of the structured domains examined here . Sequences from non-human hosts were excluded, as were sequences without a known geographic location described either in the associated literature or clearly annotated in the sequence record. Single isolate populations were also excluded. For PvDBP, bases 766–1659 were used, while for PvAMA1 bases 121–1422 were used.Reference sequences for Sal-1 oDB.org) . Genomicxi is the relative frequency of the ith haplotype and n is the number of samples.The DendroPy Python library  was usedpi is the frequency at that position of the ith amino acid in the standard 20 amino acid alphabet. The normalized Shannon entropy is a measure of sequence diversity, and takes values between 0 and 1, where 0 indicates perfect sequence conservation at that site, and 1 indicates an even distribution of all possible amino acids at that site.Normalized Shannon entropy  for eachSequences from all isolates were aligned using MUSCLE v3.8.31 , and aliA Python package, BioStructMap, which allows for the application of a 3D sliding window over a protein structure has been previously described . BioStruPvDBP structures  all contain a number of unresolved residues, and to ensure complete coverage of DBP Region II in this analysis, a template-based model of P. vivax DBP was generated for use with BioStructMap. This model was created using ModPipe [https://modbase.compbio.ucsf.edu/; ModPipe model ID f7602e019fac5be4a79c4cca6751b392) [Known  ModPipe , an auto ModPipe . The PDB751b392) .PvAMA1 model used has been previously described [PvAMA1.The escribed , and usePvAMA1 and PvDBP residue data between each pair of populations. Residues with missing data in one or both populations (i.e. Tajima’s D was undefined) were excluded from analysis for that pair of populations.To compare structural patterns of nucleotide diversity and Tajima’s D between all populations considered in this study, Spearman’s rank correlation coefficient was computed for both The majority of data analysis was performed using the Anaconda distribution of Python 3.5. Plotting was performed with the Python Matplotlib package, version 1.5.1 . StatistPvDBP and PvAMA1. Genomic sequences for each antigen were extracted from GenBank, with a total of 505 PvAMA1 sequences and 243 PvDBP sequences obtained, belonging to 10 and 12 distinct populations, respectively. Sequences which did not cover at least 95% of the structured domains examined  were excluded from analysis, as were sequences from single-isolate populations or non-human hosts. Maximum likelihood phylogenetic trees were constructed for both PvAMA1 and PvDBP , while other branches contained a mix of populations. This intermixing was particularly evident for populations that are geographically close, such as Thailand, Myanmar and Papua New Guinea for PvAMA1.This study aimed to examine selection pressures on key structured domains of PvAMA1 and PvDBP sequences were computed, as well as several measures of diversity and selection (Tables PvAMA1 (haplotype diversity = 0.99), while 84 haplotypes were observed for PvDBP RII (haplotype diversity = 0.96).Key population parameters for n Tables . A totalPvAMA1, nucleotide diversity (π) and mean number of pairwise differences (k) were highest in the three Thai populations examined, with a maximum π of 10.09 × 10−3 in the 2007 Tak Province, Thailand samples. Nucleotide diversity was lowest in the South Korean population (π = 5.90 × 10−3), which may be explained by a recent bottleneck in this population, limiting overall diversity; this is supported by the negative Tajima’s D value observed for PvAMA1 in this South Korean population (D = − 1.20). Tajima’s D is often used to identify regions under balancing selection (Tajima’s D > 0), which can be the result of immune selection pressure, and it is noted that for PvAMA1, only the Venezuelan population had a significantly positive Tajima’s D value , as per the confidence limits outlined in Tajima [For n Tajima , althougn Tajima . Most otPvDBP, nucleotide diversity was highest in South Korea (π = 10.53 × 10−3), Bangkok, Thailand (π = 10.37 × 10−3) and Myanmar (π = 10.17 × 10−3). Interestingly, the South Korean population had a negative Tajima’s D value that was statistically significant, suggestive of a recent population bottleneck and expansion. This population also had the highest number of polymorphic sites (58 non-synonymous). This high variability is probably due to 2 or 3 divergent isolates .When examining PvAMA1 as compared to PvDBP [These observations agree with previous studies that have generally observed greater signatures of immune selection pressure on to PvDBP .PvAMA1 structure both as a spatially averaged value and a per-residue value, nucleotide diversity was calculated using a 3D sliding window . A positive Tajima’s D value provides evidence for balancing selection, which can arise as a result of immune pressure. It is noted that departures from neutrality can also arise as a result of population structure. In particular, sampling of strains across multiple distinct populations can potentially give rise to similar signatures to those of balancing selection. For this reason Tajima’s D values were analysed within distinct geographic and temporal populations. Spatially-derived Tajima’s D was highest in DI for nearly all populations examined, with the exception of Sri Lanka and South Korea  had very low nucleotide diversity across all populations. Spatially derived Tajima’s D values were also calculated for PvDBP, and observed that most populations had Tajima’s D values that were negative or close to zero over most of the structure . The most frequent of these polymorphisms (N132D) is a relatively conservative change from asparagine to aspartic acid, and is the only one of the three residues whose side chains are directly involved in hydrogen bonds with RON2; the side chains from both N130 and D133 are only involved in intramolecular hydrogen bonds. It has previously been shown that a PvRON2 peptide binds well to PfAMA1 [PvAMA1 is not conserved in PfAMA1, and that this region is also not involved in the binding between PvRON2 and PfAMA1 (PDB structure 5NQF) [PvRON2 and are hence amenable to polymorphic variation.While binding interfaces were generally observed to have low nucleotide diversity, there were some polymorphic residues located within these binding interfaces for both o PfAMA1 . With thre 5NQF) . These oPvDBP dimerization and DARC binding interface, although again most of these were present at low frequencies. The highest frequency polymorphism within this region is R263S, with a minor allele frequency of 20%. In the PDB structure 4NUU, the backbone of R263 is involved in hydrogen bonds to DARC, while the side chain is involved in a single intra-molecular hydrogen bond. This residue is also part of a loop which is disordered when not bound to DARC (loop 254–267) [PvDBP and PkDBPα; PkDBPα has an arginine at this position and is also able to bind DARC, supporting the viability of the T359R mutation with regards to maintaining DARC binding ability.There were 7 polymorphic residues observed within the 254–267) , suggest254–267) , althougPvDBP RII and PvAMA1, although there were some additional regions within PvAMA1 with Tajima’s D values above the threshold for significance when using a 3D sliding window approach that were not identified with a linear sliding window. However, these regions were generally structurally contiguous with other stretches of sequence that were above the threshold for significance when using a linear sliding window.While a number of previous studies have utilized a linear sliding window approach when calculating Tajima’s D or nucleotide diversity –7, 65, tPvAMA1, and Myanmar and Papua New Guinea for PvDBP. In contrast, there was less agreement in the structural patterns of Tajima’s D over each structure. There was reasonable positive correlation between most populations for spatially derived Tajima’s D over the PvAMA1 structure, with the exception of South Korean and Sri Lankan populations. For PvDBP, the Papua New Guinean population was the major outlier when considering spatially derived Tajima’s D values, with the highest values observed on subdomain 3, furthest from the dimerization interface. The lower level of apparent immune selection pressure on PvDBP may explain the more discordant results observed between populations for PvDBP; nearly all Tajima’s D values observed for PvDBP do not meet the threshold for statistical significance (p < 0.05) as defined by Tajima [PvAMA1, including both Papua New Guinean populations  and the Venezuelan population  [Given the success using epitope focusing techniques for esponses , and thiepitopes , 67, alt1 (TfR1) .PvAMA1 and PvDBP, and it was shown that the regions of high diversity and balancing selection on the protein structure are remarkably similar across a number of populations. This suggests that dominant epitopes are the same across multiple human populations, which has positive implications for the development of a universal P. vivax vaccine. Furthermore, polymorphisms were observed to cluster around binding interfaces on both PvDBP and PvAMA1, suggesting a level of immune pressure on residues surrounding these key binding interfaces. Large regions with very low diversity were also identified for both antigens, and it is suggested that these areas may also be useful targets to focus on for further vaccine development, given previous evidence of functional antibody responses against these conserved regions.In this work, signatures of diversity and selection were identified on Additional file 1. Domains/subdomains of PvAMA1.Additional file 2. Domains/subdomains of PvDBP RII.Additional file 3. Spatially-derived nucleotide diversity for PvAMA1 across multiple populations.Additional file 4. Spatially-derived Tajima’s D for PvAMA1 across multiple populations.Additional file 5. Spatially-derived nucleotide diversity for PvDBP across multiple populations.Additional file 6. Spatially-derived Tajima’s D for PvDBP across multiple populations.Additional file 7. Location of statistically significant (p < 0.05) Tajima’s D values on modelled PvAMA1 (a) and PvDBP (b) structures.Additional file 8. Sequence diversity at interfaces.Additional file 9. Comparison of spatially derived Tajima’s D and conventional linear sliding window calculation of Tajima’s D for PvAMA1.Additional file 10. Comparison of spatially derived Tajima’s D and conventional linear sliding window calculation of Tajima’s D for PvDBP RII.Additional file 11. Table of highly polymorphic residues within PvAMA1.


Although high carbon martensitic steels are well known for their industrial utility in high abrasion and extreme operating environments, due to their hardness and strength, the compressive stability of their retained austenite, and the implications for the steels’ performance and potential uses, is not well understood. This article describes the first investigation at both the macro and nano scale of the compressive stability of retained austenite in high carbon martensitic steel. Using a combination of standard compression testing, X-ray diffraction, optical microstructure, electron backscattering diffraction imaging, electron probe micro-analysis, nano-indentation and micro-indentation measurements, we determined the mechanical stability of retained austenite and martensite in high carbon steel under compressive stress and identified the phase transformation mechanism, from the macro to the nano level. We found at the early stage of plastic deformation hexagonal close-packed (HCP) martensite formation dominates, while higher compression loads trigger body-centred tetragonal (BCT) martensite formation. The combination of this phase transformation and strain hardening led to an increase in the hardness of high carbon steel of around 30%. This comprehensive characterisation of stress induced phase transformation could enable the precise control of the microstructures of high carbon martensitic steels, and hence their properties. This work hardening may be very desirable in industrial applications in which the steel’s surface wears due to the application of stresses, but the material remains hard due to the transformation of retained austenite to martensite. Depending upon its chemical composition, retained austenite can be metastable phase and will transform to martensite by passing the phase transformation barrier energy. Martensitic transformation is achieved by the cooperative shear movement of atoms; applied compressive stress involving compression deformation aids the transformationFor many years, high carbon steels have proved useful for industrial application in extreme operation conditions due to their hardness, strength and relatively low cost compared to high alloy steels. High carbon martensitic steels are favoured when high abrasion resistance is required. These steels contain plate and lath martensite which is formed from austenite during quenching, although this transformation is rarely complete, and some austenite remains. An inverse relationship exists between the strength of the martensite formed in the quenching process and the amount of residual austenite; the martensite’s strength increases as the amount of retained austenite decreases. Hence retained austenite is generally considered deleterious in low-carbon steel1234This study focused on high carbon martensitic steel for use as a wear resistant material in industry. The mechanical stability at the micro and nano level and its stress-induced martensite transformation behaviour has been investigated by Optical microscopy, X-ray diffraction, Electron backscattering diffraction and Nano-indentation technique. In addition, the strain hardening effect on the overall hardness of the high carbon steel was investigated by micro and nano-hardness analyses. Identifying the volume percentage of each phase under compressive stress as well as phase transformation steps, is essential to characterise high carbon steel as a wear resistant material for extreme operating conditions.mm × 4 mm × 4 mm) using a diamond cutter at a very slow speed (0.01 mm/sec) to minimise both the heat effects and shear stress, thereby ensuring that retained austenite was not inadvertently transformed as the sample were prepared. Standard metallographic wet grinding and polishing methods were used to prepare the samples for X-ray analysis. A PANalytical Empyrean XRD instrument was used with unfiltered Co-Kα radiation at 45 kV and 40 mA current for quantitative XRD to measure the volume fraction of phases from a 2θ spectrum that was acquired at a step size of 0.0260 over an angular range of 40° to 130°. The compression deformation experiment was performed at room temperature with an Instron 8510 instrument operating at 0.10 mm/min cross-head speed over a loading pressure of 200 MPa to 2500 MPa.Industrial grade high-carbon steel with the chemical composition shown in ® CrossBeam® field emission gun scanning electron microscopy (FEG SEM) workstation. To determine the steel’s quantitative carbon and manganese content an electron probe micro-analysis (EPMA) was conducted using a Jeol JXA 8500F Hyper probe machine. Nano-indentation tests were carried out in load control mode on a TI 900 Hysitron Tribolab system at varying loads up to 8000 μN with a Berkovich three-sided pyramidal diamond tip indenter . As the varying loads were applied to the austenitic phase, microstructural images helped to identify the resulting phase transformation phenomena at the nanoscale. To obtain the hardness profile of the compression induced material, nano-indentations were made in a 3 × 3 matrix with a uniform load of 8000 μN. Conventional micro Vickers hardness tests were also performed to obtain macroscopic strength at 0.2 HV load.After X-ray diffraction characterization, an orientation microscopy investigation of transformed austenite and martensite was conducted by electron back-scattered diffraction (EBSD) technique, using an Oxford system attached with a Carl Zeiss AURIGAAfter heat treatment the structure of the steel was found to contain martensite and retained austenite, which were observed in the optical microscopy images in All the samples were investigated by XRD. The volume fraction of retained austenite varied within the 59–60% range in all the samples before compression testing. X-ray analysis was carried out to study the mechanical stability of the retained austenite and other phases on the macro scale under compression deformation. In the following, the volume fraction calculating methods from the XRD spectrum are described. According to the ASTM-E975–13 standard, the volume fraction of each phase can be calculated based on equation , the atomic scattering factors ki and replacements gj; p = Multiplicity factor of the (hkl) reflection; θ = Bragg angle; eM−2 = Debye-Waller or temperature factor which is a function of θ; Vi = Volume fraction of i -plane. The constant K is composed of various physical properties of the material. The terms in the R factor involve the unit cell volume, structure factor, crystallographic multiplicity factor, Lorentz polarization factor and the temperature factor.In this equation, γ), BCC-martensite (α) and HCP-martensite (ε), total volume of the phases can be written as:Therefore, for a steel containing FCC austenite γ diffraction positions. As the compressive stress was increasing the austenite peak started decreasing and the martensite peak at (110)α' started increasing. Also, the peak position shifted with the stress. These XRD phenomena correspond closely to the transformation of austenite to martensite as a result of compressive stresses. According to the literature, the peak shift occurs due to lattice expansion during the transformation of retained austenite to martensiteε and (101)ε peaks became apparent, indicating that the formation of hexagonal ε–martensite phase occurred during deformation  between 200 MPa to 1500 MPa compressive stresses. When the applied pressure exceeded a 1500 MPa load, the volume fraction of α′–martensite increased abruptly to 75%  plots2526α′ martensite phase, the locations for the nucleation of ε-martensite. This study also reveals that more ε martensite than α′ martensite forms at the beginning of the plastic deformation (up to 1500 MPa). Later, at compression stresses reach and exceed 2000 MPa α′ martensite formation was observed. These observations are made based on dislocation contents (blue zone with lower dislocation density), which are also supported by the XRD data in γ → ε transformation dominates while at the higher compression stresses γ → α′ transformation prevails.It should be noted that these EBSD scan were conducted with a pattern binding of 1 × 1, with an integration number of frames of 10 for 15 kV and that the step size chosen was less than 100 nm. These refined and high resolutions EBSD scans make it possible to detect ε-martensite and to identify the relationship between ε and −2 range. The literature shows the formation of martensite occurs below18 mJm−2 SFE283322Another factor that influences the retained austenite to martensite transformation is the stacking fault energy (SFE). The stacking fault energy is related to the C and Mn content and therefore a carbon content measurement in the retained austenite was conducted using EPMA. To observe the stress induced phase transformations EBSD scanning was carried out, before and after the nano-indentation experiments, as per the data shown in figure Broadly, the appearance of discontinuous displacement bursts in crystalline metals can be associated with certain factors, for instance; dislocation nucleation and propagation; crack formation or phase transformation3536τmax at the elastic zone can be compared with the theoretical value when the first burst occurred. This can be calculated by equation .


Metasurfaces utilizing engineered metallic nanostructures have recently emerged as an important means to manipulate the propagation of light waves in a prescribed manner. However, conventional metallic metasurfaces mainly efficiently work in the visible and near-infrared regime, and lack sufficient tunability. In this work, combining the pronounced plasmonic resonance of patterned graphene structures with a subwavelength-thick optical cavity, we propose and demonstrate novel graphene metasurfaces that manifest the potential to dynamically control the phase and amplitude of infrared light with very high efficiency. It is shown that the phase of the infrared light reflected from a simple graphene ribbon metasurface can span over almost the entire 2π range by changing the width of the graphene ribbons, while the amplitude of the reflection can be maintained at high values without significant variations. We successfully realize anomalous reflection, reflective focusing lenses, and non-diffracting Airy beams based on graphene metasurfaces. Our results open up a new paradigm of highly integrated photonic platforms for dynamic beam shaping and adaptive optics in the crucial infrared wavelength range. Metamaterials are rationally engineered composite materials with exotic properties not easily obtained or altogether unavailable in nature123568101213151718195So far, most metasurfaces rely on judiciously designed metallic nanostructures. At optical frequencies, metallic nanostructures with varied geometries support strong surface plasmon resonances, acting as local antennas in frequency selective surfaces (FSS)222325272829303132333335363839414243444546313238394048Although fairly extensive effort has been devoted to graphene-based metamaterials and plasmonics, previous work mainly focuses on the distinct resonance features and their spectral tuning. In this letter, we propose and demonstrate graphene plasmonic metasurfaces to steer infrared light with high efficiency, which drastically expand the functionalities and performance of graphene devices towards complete wave shaping. This is made possible by combining the plasmonic resonance of patterned graphene nanostructures with a subwavelength-thick optical cavity to enhance the intrinsically weak interaction between light and single-layer graphene. Furthermore, the graphene metasurfaces can be dynamically tuned by changing the Fermi energy of graphene via simple chemical or electrostatic tuning that is unachievable in conventional metasurfaces made of metals. As a proof of principle, we numerically study graphene metasurfaces consisting of rationally designed graphene ribbons. It is shown that the phase of the reflected infrared light can range almost from –π to π by tailoring the dimensions of the graphene ribbons as well as the optical cavity, while the amplitude of the reflectivity is sufficiently high without substantial variations. This allows us to design graphene metasurfaces to implement anomalous reflection, focusing and non-diffracting Airy beam generation. We envision that such transformative graphene metasurfaces would open up a new paradigm of integrated photonic systems with multifunctional applications, including dynamic beam shaping, hyperspectral imaging, adaptive optics, biomedical sensing, and energy harvesting in the critical infrared region.s of the dielectric spacing layer is smaller than the wavelength. Consequently, we can achieve strong and even perfect absorption with monolayer graphene structures as theoretically proposed and recently demonstrated in experiments525354Graphene can interact with light strongly via plasmonic resonance27383950Graphene metasurfaces coupled with an optical cavity exhibit much more striking properties and applications beyond strong absorption. Our numerical simulations show that the phase of the reflected light can vary from almost –π to π by selecting the width of the graphene ribbons as well as the thickness of the dielectric spacing layer, while the amplitude of reflectivity remains high and only shows moderate variations. These two properties allow us to design a variety of planar graphene photonic devices in the reflective configuration that are easy to fabricate and integrate. It should be emphasized that the full 2π phase coverage is crucial for designing functional metasurfaces, which necessitates the bottom metallic mirror. The bare graphene ribbons on a dielectric layer only provide a phase modulation range of about π 56, becau8913s) of the dielectric spacing layer and the width (w) of the graphene ribbons. TM polarized light can excite the plasmonic resonance of graphene ribbons. In contrast, for transverse electric (TE) polarization, the electric field is parallel to the graphene ribbons, and no plasmonic resonance can be excited to modulate the phase and amplitude of reflected light (p) of the graphene ribbon is fixed at 3 μm. In the finite element simulation utilizing commercial software COMSOL Multiphysics, the optical conductivity of graphene is described by Kubo model with the Fermi energy Ef = 0.64 eV. The refractive index of the dielectric spacing layer is taken as 1.4, and Drude model is applied to fit the experimental data of permittivity of gold for the back reflective mirrorIn this paper, we focus on the far-infrared region, while the operation wavelength of the proposed graphene metasurfaces can be readily extended to mid-infrared by scaling the dimension of the graphene patterns. ed light . The fres = 4.37 μm, the reflectivity is larger than 48.6% and the phase smoothly spans over from almost –π to π with different graphene ribbon widths as shown in From 52dφ/dx at the interface between two media. Anomalous reflection and refraction phenomena have been reported in this regime, which are in sharp contrast to the well-known refraction and reflection law8The results in ni is the refractive index of the material at the incidence side, and θi and θr are the incident and reflected angle, respectively. There is a nonlinear relation between θi and θr, distinctly different from conventional specular reflection. For normal incidence and ni = 1 (air), the reflection angle can be calculated aswhere Hy) of the reflected light. According to the field distribution, we can retrieve the reflection angle, which is about 13.1° and in excellent agreement with the predicted value. The slight deformation of the phase front arises from the finite periodicity and non-uniform reflectivity of the metasurface. If we set the incident angle as 15.0°, the reflected angle should be about 29.0°. The simulation result is shown in In our simulation, we set 36 graphene ribbons with periodicity of 3 μm. The width of each ribbon is properly selected  so that F, the phase shift distribution φ(x) along the interface should satisfyThe second graphene metasurface device to be demonstrated is a reflective focusing lens. To design such a lens, we must ensure that the light reflected from every graphene ribbon has the same phase or with 2π multiplied by an arbitrary integer when propagating to the focal point. In other words, the metasurface should carry a phase profile to compensate the propagation phase between graphene ribbons and the focal point, so that the radiated waves from the metasurface can constructively interfere with each other at the focal point. For a certain focal length F = 100 μm, and x = mp with p equal to the periodicity of the graphene ribbons and m = 0, ±1, ±2… From the results presented in x axis  and w2 = 1058 nm  to match the simplified profile  planar systems59 profile . From FiWe have introduced graphene metasurfaces based on simple ribbon geometries to efficiently steer infrared light in the reflective configuration. The graphene ribbons coupled with a subwavelength-thick optical cavity substantially enhance the light-graphene interaction, allowing us to achieve almost 2π phase modulation and high reflectivity amplitude. Anomalous reflection, reflective focusing lenses and non-diffracting beam generation with capability of dynamic tuning have been successfully demonstrated. Although we have focused on the far-infrared frequency, the design strategy can be readily applied to mid-infrared region by varying the dimension of graphene ribbons and thus their plasmonic resonance. Moreover, extending ribbons to other 2D patterns provides additional degrees of freedom in the design, promising other important advantages for light steering such as 3D focusing, polarization-independent functionalities, and strong polarization conversion. Leveraging the remarkable plasmonic properties of graphene at infrared wavelengths, the graphene metasurfaces demonstrated here manifest unprecedented control over the electromagnetic properties of materials on a completely 2D platform, which enable disruptive light-weight, ultra-compact, and high-speed optical architectures for diverse applications.In the terahertz and infrared region, the optical conductivity of graphene is dominated by the intraband transition. It can be described by a semi-classical Drude modelkB is the Boltzmann constant, Τ is the temperature, Ef is the absolute value of the Fermi energy, and τ is the electron relaxation time. When the Fermi energy Ef is much larger than the thermal energy kBT, the conductivity of graphene can be further simplified to τ is related to the carrier mobility μ, the Fermi energy Ef and Fermi velocity vf as τ = μEf/evf2, which indicates that increasing carrier mobility of graphene will reduce the loss and improve the device efficiency. The carrier mobility μ of graphene film ranges from ∼1000 cm2/Vs in chemical vapor deposition (CVD)-grown graphene to 230,000 cm2/Vs in suspended exfoliated graphene. In our simulations, we assume the Fermi energy of graphene Ef around 0.64 eV, a moderate mobility μ = 10,000 cm2/Vs, Fermi velocity vf = 106 m/s and scattering time τ = 6.4 × 10−13 s27Ef = 1–2 eV has been reported652/Vs has been achieved by using a hexagonal boron nitride substrate even with thickness as small as 14 nm67σv = σ/t assuming that t is a very small value2), Calcium Fluoride (CaF2), or Magnesium Fluoride (MgF2), is employed in the design as the dielectric spacing layer, which is assumed to have a refractive index of 1.4.where How to cite this article: Li, Z. et al. Graphene Plasmonic Metasurfaces to Steer Infrared Light. Sci. Rep.5, 12423; doi: 10.1038/srep12423 (2015).


Our primary outcome was change in cerebral vessel diameter and the secondary outcome was change in neurobehavioral scores. There was a 40.8 ± 8.2% increase in cerebral vessel diameter in those animals treated with αCGRP compared with controls . Neurobehavioral scores were reported in four publications and showed a standardized mean difference of 1.31 in favor of αCGRP (CI −0.49 to 3.12). We conclude that αCGRP reduces cerebral vessel narrowing seen after SAH in animal studies but note that there is insufficient evidence to determine its effect on functional outcomes.Delayed cerebral ischemia (DCI) is a life-threatening complication after subarachnoid hemorrhage. There is a strong association between cerebral vessel narrowing and DCI. Alpha calcitonin gene-related peptide (αCGRP) is a potent vasodilator, which may be effective at reducing cerebral vessel narrowing after subarachnoid hemorrhage (SAH). Here, we report a meta-analysis of data from nine  Aneurysmal subarachnoid hemorrhage is a significant cause of morbidity and mortality worldwide with an annual incidence of 9 per 100,000 person years . 27% of Despite successful treatment of the ruptured aneurysm and reduction of the risk of rebleeding, approximately 30% of treated patients develop focal neurological or cognitive deficits conventionally attributed to delayed cerebral ischemia (DCI) –11. A stAlpha calcitonin gene-related peptide (αCGRP) is an endogenous neuropeptide and powerful vasodilator. αCGRP exerts its relaxing properties through nitric oxide and endothelium-dependent or endothelin-independent pathways . Endogenin vivo animal studies that investigated the effects of αCGRP after experimental SAH.Here, we use this approach to summarize data from publications reporting The study protocol has been published elsewhere and is available open access but a brief description is given below .via PubMed Central, and EMBASE via OvidSP) using the key words “alpha calcitonin gene-related peptide,” “αCGRP,” and “subarachnoid hemorrhage” in combination using the Boolean operator [AND]. The search was restricted to “other animals.” Two investigators (Liam M. C. Flynn and Caroline J. Begg) independently screened the abstracts and titles to identify those that met our inclusion criteria. Any differences were resolved by discussion with a third reviewer (Peter J. D. Andrews). We included in vivo animal studies describing the effect of αCGRP in animal models of SAH where outcome was reported as a change in arterial diameter and the articles were published in English. We also included studies which examined in vivo SAH and αCGRP administration with postmortem in vitro measurements of artery diameter.In January 2015, we searched two electronic databases ; anesthetic agent used; dose of αCGRP and time of administration from SAH; reporting and method of randomization; reporting and method of blinding; animal welfare guideline statement; statement of sample size calculation; whether there was a statement of potential conflicts of interest and the use of animals with comorbidities. Study quality was assessed using the CAMARADES 10-item quality checklist  statement of compliance with regulatory requirements, (9) conflicts of interest statement, and (10) use of animals with comorbidities.Two investigators independently extracted data relating to species of animal and weight; method of inducing SAH ; whether the basilar artery, middle cerebral artery, anterior cerebral artery, or internal carotid artery were measured; the method of measurement . Where a single control group was used for multiple treatment groups, we adjusted the size of the control group entered into the meta-analysis by dividing the size of the control group by the number of treatment groups served .Two of the nine publications did not report quantitative data in their text, only presenting graphed data , 32. Meain vivo experiments investigating the effect of αCGRP in animal models of SAH where outcome was reported as a change in arterial diameter were included. We also included experiments which examined in vivo SAH and αCGRP administration with postmortem in vitro measurements of artery diameter. Where one study administered different doses of αCGRP to separate groups of animals but had a shared control group we treated these as separate experiments and divided the number of controls between the experiments as per Vesterinen et al.  and those which lacked SAH models, nine eligible publications remained . Four experiments used New Zealand White, and eight experiments used Japanese White. Five experiments used mongrel dogs . Two experiments used rats . One used Wistar rats, and the other used Sprague Dawley rats. The remaining experiment used Macaca fascicularis . The median number of study quality checklist items scored was 4 (IQR 2–6). No studies used animals with comorbidities; reported a statement of potential conflicts of interest or stated an a priori sample size calculation. 40% of studies reported control of body temperature. 20% described a randomized treatment allocation, and 45% reported allocation concealment. Half of the experiments used a blinded assessment of outcome, and half used an anesthetic agent other than ketamine. All studies were published in peer-reviewed journals, and 70% reported compliance with local animal welfare guidelines. In 16/20 (80%) of the experiments, SAH was induced by autologous blood injection by either single or double injection methods, the remainder were induced with blood clot placement. Further study characteristics are presented in Tables The total number of animals included in analysis was 251, and the median number of animals used per experiment was 9 [interquartile range (IQR) 6–14]. Twelve experiments used rabbits  included in meta-analysis, there was a 40.8 ± 8.2% increase in cerebral vessel diameter in the αCGRP group compared with controls  from experiments with a quality score (QS) of 1 to 28.1 ± 9.1% (p < 0.01) from experiments with a QS of 6  in favor of αCGRP. Tian et al. reported neurological outcome based on a comprehensive scoring system  measured three times daily and based upon the assessment of four functions, which has been used elsewhere (p < 0.001). Imaizumi et al. assessed neurological outcome based on food intake and a slope tolerance test on days 2, 3, and 4 post-SAH  that pharmaceutical interventions have decreased the incidence of radiographic vasospasm  without decreasing poor clinical outcomes . The autThe CONSCIOUS trials investigated the effect of the endothelin-receptor antagonist (ERA), Clazosentan, in the treatment of DCI after SAH , 52. ClaThere were no female-only experiments and the majority of experiments in this systematic review used the single hemorrhage model of SAH (60%). The other forms were double injection and clot placement. Animals rarely develop a vessel narrowing-related ischemic neurological deficit from any of these methods. Megyesi et al. note that this is probably because animal brains have a plentiful collateral blood supply . While tvia gene transfer and the other by intranasal delivery. If intraventricular administration of αCGRP in humans ameliorates cerebral vessel narrowing and avoids the adverse effects seen with intravenous delivery, both gene transfer and intranasal delivery offer potential alternatives without the difficulties associated with an intraventricular drain.One of the challenges of translating these animal data and methods to human trials is the invasive nature of the intrathecal route. Previous human trials with intravenous administration of αCGRP have used a continuous infusion owing to the short half-life of αCGPR in the systemic circulation (approximately 7–10 min) . HoweverWe demonstrate a significant dilation of cerebral arteries after αCGRP administration in animal models of SAH. However, there is insufficient animal data to determine the effect of αCGRP on neurobehavioral outcomes after SAH. We recommend that any future experimental studies investigating the effect of αCGRP in SAH include neurobehavioral scores as an outcome measure. The dilatory effect of αCGRP appears augmented after SAH, and there is some evidence that systemic effects of αCGRP are lessened by intrathecal administration compared with intravenous administration.LF and CB conducted independent literature searches and performed dual data entry. LF wrote the manuscript. PA and MM edited the manuscript and provided input into review design. MM performed statistical analysis.LF and PA have previously applied to the Medical Research Council for funding for a clinical trial involving the administration of αCGRP to patients who have suffered an aSAH.


Then, we explored the potential interaction between Y-hg O3e* and autosomal variants. Our results demonstrated that there was a suggestively significant interaction between Y-hg O3e* and rs11135484 on NOA (Pinter = 9.89 × 10−5). Bioinformatic analysis revealed that genes annotated by significant single nucleotide polymorphisms (SNPs) were mainly enriched in immunological pathways. This is the first study of interactions between Y-hgs and autosomal variants on a genome-wide scale, which addresses the missing heritability in spermatogenic impairment and sheds new light on the pathogenesis of male infertility.The Y chromosome has high genetic variability with low rates of parallel and back mutations, which make up the most informative haplotyping system. To examine whether Y chromosome haplogroups (Y-hgs) could modify the effects of autosomal variants on non-obstructive azoospermia (NOA), based on our previous genome-wide association study (GWAS), we conducted a genetic interaction analysis in GWAS subjects. Logistic regression analysis demonstrated a protective effect of Y-hg O3e Mutagenesis studies in vivo have identified hundreds of potential causal genes that impact the process of reproduction3Infertility is the inability of a sexually active couple to achieve pregnancy without contraception in one yearAmong these genetic studies, Genome-wide association studies (GWAS) present a powerful tool to detect candidate genes for a trait. Our previous GWAS identified some susceptibility loci for non-obstructive azoospermia (NOA) in Han Chinese68Y chromosome, in contrast to the rest of the genome, is confined to males and contains the smallest number of genes, most of which locate in the male specific region (MSY)1213In fact, as a mainly genetic background, Y-hgs may underlie phenotypic variations like SNPs in autosomes. And association studies considering only one of the Y chromosme or autosomes are likely to attain inconsistent results among different human populations or even in the same population1516P = 5.55 × 10−3). On the contrary, frequencies of Y-hgs O3e1 and O2* was higher in the case group  than those in the control group , although there were no significant differences between these two groups.To assess whether some Y-hgs are predisposing to or protecting against the spermatogenic impairment, we compared the distributions of Y-hgs between cases and controls. As shown in Pinter ≤ 1 × 10−4) and presented in P = 9.09 × 10−4 and executed a beneficial effect on azoospermia (ORSNP = 0.80). Interaction between Y-hg O3e* and rs11135484 was synergistic (ORinter = 2.07). Besides, rs17217643, rs6774209, rs11135482, rs17139327, rs4757259 and rs8035166 were also protective factors which magnified their effects on the presence of Y-hg O3e*. On the contrary, rs11678378, rs12520985, rs9452333 and rs9510242 showed antagonistic interactions with Y-hg O3e*.From the first-step screening, there was only one significant Y-hg O3e* associated with NOA. Thus, in order to examine potential modifications of the Y-hg on effects of SNPs, we further compared the distribution of SNPs among NOA cases and controls based on the frequency of Y-hg O3e*. Although there is no demonstration of a statistically significant result here, however, totally 38 Y-hg O3e*-SNP pairs were found to be suggestively significant (ERAP2 (P = 5.1 × 10−32 for both).To examine whether SNPs in In order to explore potential functions of SNPs, we performed pathway analysis using gene ontology (GO) enrichment analysis. As presented in GWAS has become a powerful tool for genetic scientists in the past 10 years. The strategy of GWAS is to uncover the SNPs which occur differently in people with or without a particular disease like cancer, Alzheimer disease, obesity, etc. Using this solution, we have identified several potential risk genomic regions of NOA in our previous studies6The X and Y chromosomes, the sex chromosomes, are special for men since the hemizygous exposure. While the abnormalities in the X chromosome are reported to be associated with a much wider range of diseases, the Y chromosome is believed to play a pivotal role in sex determination and spermatogenesis. The deletions of the AZF region in the Y chromosome long arm lead to spermatogenic impairment22So in this study, we hypothesized that individuals in predisposed Y-hgs may carry some autosomal variants, which might be a potential genetic modifier for the Y-hg specific susceptibility to spermatogenic impairment. We first examined whether some Y-hgs are risk or protective factors for spermatogenic impairment. Our results demonstrated that Y-hg O3e* was significantly associated with NOA. Compared with the reference Y-hg O3*, Y-hg O3e* was protective against the prevalence of spermatogenic impairment. The diversity of haplogroups is the result of genetic drift, natural selection or stochastic processes, and the beneficial effect on NOA cannot be occasional. Although the machanisms by which haplogroups exert their functions are unclear, emerging evidence has been reported to connect different haplogroups with variations of phenotypes, including high or reduced sperm motility2, which was a measure of goodness of fit of the statistical model, was also shown. Although pseudo-R2 values were somewhat low here, they were still helpful in the model building state as a statistic to evaluate competing models and might be quoteworthy for other researchers on the male infertility. Besides that, we found that rs11135484 was suggestive of an interaction with Y-hg O3e* on NOA. Rs11135484 locates in an intron of ERAP2, which was found in the cytoplasm/endoplasmic reticulum (ER) and the plasma membrane. ERAP2 is a proteolytic enzyme site in the ER where it plays a central role in antigen processing and presentationERAP2 can affect its nonsense-mediated RNA decay and protein expression25ERAP2 which means they can regulate the expression of ERAP2. Moreover, the Encode project also points out that rs11135484 located in a region marked by enhancer markers, and it may change transcription factors binding affinity resulting in the different expression of ERAP2. In addition, a possible effect of ERAP2 deficiency could be an alteration in a quantitative reduction of MHC levels and thus influence the homeostasis of reproductive functionNext, to find out the autosomal variants with which Y-hg O3e* might interact, we combined data from Y-hgs with those from our previous GWA study. The pseudo-Rin vivo fertilization, spontaneous abortions etc28Pathway enrichment analysis showed that some processes like “natural killer cell activation” and “antigen processing and presentation of peptide antigen via MHC class I” shared common genes involved in immunology. For all we know, immunology is an important biological process that deals with the response of body to exo- or endo- disturbance, and many studies have linked it to the reproductive problems such as infertility, failed In summary, we combined Y-hgs with GWAS to investigate potential interactions between them on NOA, and observed that Y-hg O3e* may modify the risk of some SNPs. These results suggest that both the Y chromosome and autosomes may jointly contribute to the reproductive outcomes. We cannot always divide them into two distinct aspects, and the combination may shed new light on the pathogenesis of male infertility.This study was approved by the ethics review board of Nanjing Medical University, and all experimental protocol were in accordance with guidelines approved by the Institutional Review Board for Human Studies of Nanjing Medical University. Totally, 1,000 NOA cases and 1,703 male controls included in this study were reported in our previous genome-wide association studyY chromosome haplogtyping was performed in all 2703 participants. Totally, 10 highly informative polymorphic loci for East Asians  were identified following the nomenclature recommended by the Y Chromosome Consortium (YCC) and its updates3132P < 1 × 10−5). Individuals with overall genotype completion rates <95%, gender discrepancies, unexpected duplicates or probable relatives, heterozygosity rates >6 s.d. away from the mean or outliers in the principal component analysis were also excluded. After quality control processing, a total of 957 NOA cases and 1634 healthy controls with 587347 SNPs were included in the subsequent interaction analysis.The GWAS was conducted using an Affymetrix Genome-Wide Human SNP Array 6.0 followed by a quality control procedure as described previously0 + β1 × Y-hg + β2 × SNP + β3 × (Y-hg × SNP). Here, Y is the logit of the probability of being an infertile case, β0 is a constant, β1 and β2 are the main effects of Y-hg and SNP, respectively, and β3 is the interaction parameter to be tested. P value ≤ 1 × 10−8 was regarded as statistically significant in interaction analyses considering the issue of multiple comparison, and P value ≤ 1 × 10−4 was suggestive of an interaction.Statistical analyses were performed using R software . Firstly, distributions of Y-hg among cases and controls were assessed, and the statistically significant Y-hg was further combined with SNPs. For the analysis of the Y-hg × SNP interaction, we firstly defined the Y-hg variable as Y-hg O3e* (1) or non-Y-hg O3e* (0). And SNPs were coded as continuous variables  under an additive genetic model. Then we tested the interaction between each pair of Y-hg and SNP by conducting a 1-degree-of-freedom Wald test of a single interaction term as implemented in an unconditional logistic regression based on the equation Y = βhttp://geneontology.org/) was performed to examine biological processes encompassing statistically significant genes involved in our study. In addition, using publicly available data from the Genotype-Tissue Expression (GTEx) eQTL Browser , we examined cis associations between SNPs and expression of nearby genes in different tissues.GO enrichment analysis .