RNAs (miRNAs) are confirmed to be tumor promoters or suppressors in multiple squamous cell carcinomas (SCCs). miR‐99a‐5p has been demonstrated to be downregulated in cancerous tissues, but its functional role in head and neck SCC (HNSCC) and its mechanism of action have not been fully elucidated. Here, we studied the expression of miR‐99a‐5p in HNSCC and performed a clinical value assessment and then extracted mature expression data from The Cancer Genome Atlas (TCGA) and microarrays from Gene Expression Omnibus (GEO). Furthermore, biological analysis was constructed via online prediction tools. The results revealed that miR‐99a‐5p expression was markedly lower in HNSCC tissues than in normal tissues, which also showed significance in the prognosis of HNSCC. However, its diagnostic value could not be verified due to the lack of body fluid samples. Additionally, miR‐99a‐5p was expressed at higher levels in patients with low histological grade neoplasms than those with high histological grade neoplasms. The age of the patient might also be a possible clinical parameter affecting miR‐99a‐5p expression. Furthermore, miR‐99a‐5p significantly influenced HNSCC progression by regulating the PI3K‐Akt signaling pathway, in which the key target genes were upregulated in 519 HNSCC tissues compared to 44 normal tissues, as determined by the Gene Expression Profiling Interactive Analysis (GEPIA). In conclusion, our study may provide insights into the expression and mechanism of miR‐99a‐5p in HNSCC. Further studies are required to elucidate the role of miR‐99a‐5p and its potential clinical applications for HNSCC.Micro BPbiological processCCcellular componentDAVIDDatabase for Annotation, Visualization, and Integrated DiscoveryFNfalse negativeFPfalse positiveGEOGene Expression OmnibusGEPIAGene Expression Profiling Interactive AnalysisGOgene ontologyHNSCChead and neck squamous cell carcinomaIGF1Rinsulin‐like growth factor 1 receptorKEGGKyoto Encyclopedia of Genes and GenomesLRlikelihood ratioMFmolecular functionMTORmechanistic target of rapamycinPDGFRBplatelet‐derived growth factor receptor, beta polypeptidePIK3CDphosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit dataPPIprotein–protein interactionROCreceiver operating characteristicSCCsquamous cell carcinomaTCGAThe Cancer Genome AtlasTNtrue negativeTNMtumor, node, and metastasisTPtrue positiveSquamous cell carcinomas (SCCs), also known as epidermoid carcinomas, are cancers that derived from squamous epithelial cells, which occur in the head and neck, thyroid, esophagus, lung, penis, prostate, bladder, vagina, and cervix A deeper understanding of HNSCC is accompanied with some remarkable explorations for diagnosis, prognosis, and potential pathogenesis MicroRNAs (miRNAs) are small noncoding RNAs with 21–25 nucleotides, which have been confirmed to be involved in the initiation and development of multiple SCCs In HNSCCs, miR‐99a‐5p has been reported to be downregulated in cancerous tissues http://xena.ucsc.edu/; Accession Number: MIMAT0000097). The IIIuminaHiseq platform included 483 HNSCC patients and 44 adjacent noncancerous head and neck tissues, while the IIIuminaGA platform included 36 patients with HNSCC. No further transformation was performed for the expression data. We explored the possible association between miR‐99a‐5p expression and clinical parameters for HNSCC patients using the two platforms together or the IIIuminaHiseq platform alone, for further comparison of these two approaches. Based on TCGA data, the diagnostic and prognostic significance of miR‐99a‐5p was evaluated using the receiver operating characteristic (ROC) curve and the Kaplan–Meier curve, respectively.Mature expression data of miR‐99a‐5p in HNSCC and clinical information were obtained from TCGA datasets via UCSC (http://www.ncbi.nlm.nih.gov/geo/) and ArrayExpress (https://www.ebi.ac.uk/arrayexpress/). The search terms were as follows:  AND . Microarray datasets were eligible with the entry criteria listed below: (1) Patients in each dataset were diagnosed with HNSCC; (2) both cancerous and noncancerous specimens were included in each dataset with a sample size of no less than three per group; and (3) miR‐99a‐5p expression data should be provided. Several relevant elements were extracted from the microarray datasets: author, publication year, country, platform, sample size, and miR‐99a‐5p expression level. Two authors (Yu‐ting Chen and Jianni Yao) independently extracted essential information from all selected chips. Conflicting opinions were solved by a discussion.We obtained available miRNA expression profiling of HNSCC from the GEO database  AND  AND . Studies that provided case numbers, mean, and standard deviation (SD) were included.I2 tests. Sensitivity analysis was added to explain the heterogeneity. Results were considered statistically significant if the observed SMD with 95%CI did not cross 0. Additionally, we constructed Begg's funnel and Egger's plot to detect publication bias.Statistical analyses were performed using SPSS 23.0  and Stata version 12.0. Scatter diagrams were plotted for each study using GraphPad Prism 7.0. We also used SPSS 23.0 to calculate the mean ± SD for all the studies based on the expression value of miR‐99a‐5p. Stata version 12.0 was used to perform continuous variable meta‐analysis by evaluating the overall SMD and 95% CI. Both fixed‐effect and random‐effect model were employed, while the heterogeneity was analyzed by chi‐square and For diagnostic tests, we used SPSS 23.0 to plot the ROC curve and to calculate the true positive (TP), false positive (FP), false negative (FN), and true negative (TN) for each included study. Then, diagnosis meta‐analysis was performed via MetaDisc 1.4. Sensitivity, specificity, positive likelihood ratio (+LR), negative likelihood ratio (−LR), and diagnostic odds ratio (OR), as well as the summarized ROC curve (SROC), were chosen to describe the possible diagnostic value of miR‐99a‐5p for HNSCC. For practical application, we made a conclusion via the overall consideration of our diagnosis test results and the provided body fluid samples.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE85614 (log2FC < 0), TCGA database (log2FC > 1 and P < 0.05). The miRwalk 2.0, which included miRWalk, Targetscan, miRanda, miRDB, miRNAMap, miRBridge, RNA22, miRMap, PITA, RNAhybrid, PicTar, and Microt4, was also applied to selected genes with a computer algorithm. Genes overlapping at least two prediction platforms were selected. Based on the above source, prospective genes were screened through intersection by online tools (http://bioinformatics.psb.ugent.be/webtools/Venn/). Meanwhile, validated genes from publications were also added.To predict the putative target genes of miR‐99a‐5p, we acquired candidate genes from https://david.ncifcrf.gov/) to determine the underlying mechanism of miR‐99a‐5p in HNSCC. The STRING database (https://string-db.org/) was also utilized to construct a PPI network for further characterizing the interactions among promising target genes of miR‐99a‐5p. Furthermore, hub genes with over five degrees were selected. In addition, we acquired differentially expressed genes of HNSCC from the Gene Expression Profiling Interactive Analysis (GEPIA)  and conducted another KEGG pathway analysis to detect the potential pathways for the progression of HNSCC.Based on the predicted target genes, we conducted Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using online tools (Based on GEPIA P < 0.001). In addition, miR‐99a‐5p was expressed at higher levels in G1–G2 than in G3–G4 neoplasms . When statistical analysis was carried out using a combination of the IIIuminaHiseq and IIIuminaGA platforms (Table P < 0.001). Significant differences were also observed among neoplasms of different histological grades . In addition, miR‐99a‐5p expression was higher in patients over 50 years than in those less than 50 years . As for the diagnostic test based on TCGA, miR‐99a‐5p might show significant diagnostic value for HNSCC  and random‐effect  models  and a specificity of 0.85 . Furthermore, likelihood ratios were calculated , respectively. The diagnostic OR of 8.23  also suggested a significant diagnostic value when based on all the included samples. However, only the http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41268 and http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69002 chips provided body fluid samples . Thus, the clinical diagnostic value of miR‐99a‐5p for HNSCC could not be fully verified.ROC curves for all eligible studies were plotted. Four representative ROC curves including MTMR3, IGF1R, MTOR, NOX4, and HOXA1, which were searched from published literature, were also included We screened 14 174 genes from GSM2279805, 1532 genes from the TCGA dataset, and 3085 genes from miRwalk 2.0 after removing duplicates. As the analytical integration shown, a total of 98 genes overlapped in the GSM microarray and online software Fig. . MTMR3, P < 0.05), including cell migration and phosphatidylinositol‐mediated signaling in BP, and focal adhesion in CC. Additionally, KEGG pathway analysis also indicated that the PI3K‐Akt signaling pathway and pathways in cancer were the most enriched for miR‐99a‐5p in HNSCC , cellular component (CC), and molecular function (MF). The potential target genes of miR‐99a‐5p significantly influence 15 GO terms .The 103 putative target genes were inputted into STRING for constructing a PPI network Fig. . There wPIK3CD, IGF1R, PDGFRB, and MTOR all exhibited higher expression levels in 519 HNSCC tissues compared to the 44 normal tissues. PIK3D and IGFR1 expression levels were significant negatively correlated to miR‐99a‐5p in HNSCC , while PDGFRB and MTOR were mildly negatively correlated with miR‐99a‐5p  expression then suppress the metastasis of oral cancer (OC) et al. also reported that IGFR1 and MTOR were repressed by ectopic transfection of miR‐99‐5p et al. suggested that downregulation of miR‐99a‐5p contributed to oral squamous cell carcinoma (OSCC) by targeting MTOR, further indicating the relation between miR‐99a‐5p and HNSCC development PIK3CD), insulin‐like growth factor 1 receptor (IGFR1), platelet‐derived growth factor receptor, beta polypeptide (PDGFRB), and mechanistic target of rapamycin (MTOR) were involved.Several studies have demonstrated that miR‐99a‐5p could affect proliferation, migration, and invasion in various cancers, including HNSCCs, via modulating gene expression PIK3CD, IGFR1, PDGFRB, and MTOR4, also screened out by PPI construction, were further utilized to analyze the correlations with miR‐99a‐5p. We found that the four hub genes all exhibited higher expression levels in HNSCC tissues than in normal tissues, gaining more possibility to be the target genes of miR‐99a‐5p. Interestingly, a negative correlation was found between these four genes and miR‐99a‐5p, with PIK3D and IGFR1 showing significant negative correlation with miR‐99a‐5p in HNSCC and PDGFRB and MTOR showing a mild negative correlation. Thus, together with the findings of previous researches and our results, we speculated that the dysfunctional PI3K‐Akt signaling pathway was implicated in the development of HNSCC. Moreover, it seems that PI3K‐Akt signaling pathway was regulated by miR‐99a‐5p according to the statistical correlation analysis, which further provided evidence for the potential clinical value of miR‐99a‐5p detection in HNSCC.The PI3K‐Akt signaling pathway, concretely explained as the phosphatidylinositol 3‐kinase (PI3K)/AKT/mammalian target of the rapamycin (mTOR) signaling pathway, is aberrant in many types of cancer Statistical analysis of miR‐99a‐5p expression would confirm our speculation. Previous studies have suggested that repressed miR‐99a‐5p may contribute to tumorigenesis via being unable to control the target genes. Thus far, no study has specifically analyzed the miR‐99a‐5p expression level in HNSCC, but several studies have demonstrated the lower expression of miR‐99a‐5p in HNSCC PIK3CD, IGFR1, PNGFRB, and MTOR.This study has some limitations. First, the different miRNA extraction methods may disturb the results of our meta‐analysis. Second, the HNSCC and corresponding samples were mostly derived from tissue sections, lead to an unverified diagnostic value. Third, although we utilized TCGA data to expand our data, the big gap between the quantity of cancerous and noncancerous tissues brought down the reliability. And fourth, the resource of online protein databases and relevant immunohistochemical staining samples were limited so that we could not further validate the function of miR‐99a‐5p via the protein level of hub genes. Despite these limitations, based on our meta‐analysis, the results suggest that miR‐99a‐5p expression was significantly lower in HNSCC than in normal tissue. Biological analysis also suggested that miR‐99a‐5p may participate in HNSCC by suppressing the hub genes, In general, our study confirmed that miR‐99a‐5p might be a tumor suppressor in HNSCC with downregulated expression in HNSCC tissues, via the PI3K‐Akt signaling pathway. Further studies are required to elucidate the role of miR‐99a‐5p in diagnosis and prognosis for HNSCC and provide the basis for that miR‐99a‐5p execute its function via the protein level of the targets, especially those hub genes predicted by our research.YQ, KH, FW, and YF conceived and designed the study. YC and JY collected, extracted, and analyzed the data. YQ and KH guided the statistical process and ensured the necessary graphs. YC and JY wrote the manuscript. All authors read and approved the final manuscript.Fig. S1. (A‐K): Scatter plots of miR‐99a‐5p expression data in normal and HNSCC tissues in the other 11 microarrays without statistical significance. (L‐X): ROC curves of the other 13 microarrays without statistical significance.Click here for additional data file.


We report the migration of SugarQb to the latest and free version of Proteome Discoverer 2.1, and apply it to the analysis of PNGase F‐resistant N‐glycopeptides from mouse embryonic stem cells. The analysis of intact glycopeptides highlights unexpected technical limitations to PNGase F‐dependent glycoproteomic workflows at the proteome level, and warrants a critical reinterpretation of seminal datasets in the context of N‐glycosylation‐site prediction.SugarQb ( Glycosylation, the covalent attachment of simple or complex carbohydrate structures onto proteins, is one of the most abundant post‐translational modification (PTM), and affects virtually all aspects of life.Consequently, pioneering studies in the field of glycoproteomics primarily focused on the identification of enzymatically de‐N‐glycosylated peptides by MS/MS.Although these seminal studies were intrinsically limited to the analysis of enzymatically de‐N‐glycosylated peptides, they greatly contributed to our current knowledge of N‐glycosylation site occupancy within the proteome and provided the basis for many advanced N‐glycosylation‐site prediction algorithms. For example, using this approach in a single large‐scale study, more than 6000 N‐glycosylation sites within the murine proteome have been mapped site specifically.http://www.imba.oeaw.ac.at/sugarqb) analyses MS/MS spectra for the presence of potential [peptide + HexNAc]+ fragment ions. For this, the mass of the respective precursor ion is iteratively reduced by the masses represented in a user‐defined glycan mass database , −203.0794 amu. In cases where a corresponding potential [peptide + HexNAc]+ fragment ion is detected the respective spectra are duplicated, with the original precursor ion mass being set to the mass of the potential [peptide + HexNAc]+ fragment ion. Subsequently, the preprocessed MS/MS spectra are searched using commonly used MS/MS search engines for peptide sequence identification. In addition to this core functionality, SugarQb also provides a range of other computational tools for the automated identification of glycopeptide MS/MS spectra , charge deconvolution, and de‐isotoping , as well as the specific removal of highly abundant glycan‐derived fragment ions . SugarQb is freely available as Node to the Proteome Discoverer Platform and thus readily integrated into typical shot gun proteomic data interpretation workflows. It allows for taking advantage of modern MS instrumentation , quantitative proteomic tools , and the retrospective analysis of untargeted MS/MS datasets. Additionally, we now also migrated SugarQb to the new, freely available Proteome Discoverer 2.1 platform .Aiming at a comprehensive characterization of intact glycopeptides from complex samples, we recently developed a collection of data interpretation tools, which allows for the automated identification of intact glycopeptides from high‐resolution HCD MS/MS datasets, using well‐established proteomic MS/MS search engines  per milligram peptide in 200 mM Tris/HCl, pH 8.0, at 37 °C for 18 h. Then, we enriched the remaining glycopeptides using IP‐HILIC and analyzed them by RP‐nLC‐ESI‐MS/MSTo identify potentially PNGase F‐resistant N‐glycopeptides, we first generated tryptic digests from whole cell lysates of mouse embryonic stem cells (mESC), desalted them using SPE C18 cartridges and subjected them to enzymatic de‐glycosylation by incubation with 1 U of PNGase F . Surprisingly, despite extensive enzymatic de‐N‐glycosylation of the samples, next to 183 O‐glycosylated glycopeptides, we also identified the amino acid sequences of 186 “PNGase F‐resistant,” intact N‐glycopeptides, using MASCOT. Of note, this compares to 1110 N‐glycopeptide sequences identified by MASCOT in the control samples, which were not treated with PNGase F . MS Amanda was performed similarly . Detailed amino acid sequence analysis of “PNGase F‐resistant” tryptic N‐glycopeptides  for 18 h at 37 °C. After incubation, the individual samples were adjusted to pH 2 by the addition of 10% formic acid, pooled and desalted using SPE C18 cartridges. Eventually PNGase F‐resistant peptides were enriched using IP‐HILIC . In contrast to the previous, TMT label‐free experiment, we did not observe an important resistance of TMT‐labeled N‐terminally N‐glycosylated peptides. Indeed, 5 U PNGase F mgt Figure B. In conFurthermore, the quantitative glycoproteomic data highlighted N‐glycosylated asparagine residues, which lie at the penultimate position of tryptic glycopeptides, to be poor substrates for PNGase F , to be highly resistant to the deglycosylation by PNGase F. This observation sheds new light on previously published glycoproteomics data. For example, Zielinska et al.,In summary, we here report on the analysis of intact N‐glycopeptides from mESC, which are insensitive to the enzymatic deglycosylation with different concentrations of PNGase F. Our analyses were performed using the recently developed SugarQb platform within the freely available Proteome Discoverer 2.1 environment. The results of our analysis of intact glycopeptides highlight subtle technical limitations intrinsic to PNGase F‐dependent glycoproteomic workflows at the proteome level, and warrant a reinterpretation of these seminal datasets in the context of N‐glycosylation site prediction.The authors declare no conflict of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


Using voxelwise Spearman correlations, we tested the correspondence of methods throughout the brain. All four methods showed associations that varied across tissue types; the highest correlations were found between MWF‐D and qT1 (median ρ across tissue classes 0.8) and MWF‐G and MWF‐D (median ρ = 0.59). In eight WM tracts, all measures showed differences (p < 0.05) between MS normal‐appearing WM and healthy control WM, with qT1 showing the highest number of different regions (8), followed by MWF‐D and MTR (6), and MWF‐G (n = 4). Comparing the methods in terms of their statistical sensitivity to MS lesions in WM, MWF‐D demonstrated the best accuracy . To aid future power analysis, we provide the average and standard deviation volumes of the four techniques, estimated from the healthy control sample.Quantitative magnetic resonance imaging (MRI) techniques have been developed as imaging biomarkers, aiming to improve the specificity of MRI to underlying pathology compared to conventional weighted MRI. For assessing the integrity of white matter (WM), myelin, in particular, several techniques have been proposed and investigated individually. However, comparisons between these methods are lacking. In this study, we compared four established myelin‐sensitive MRI techniques in 56 patients with relapsing–remitting multiple sclerosis (MS) and 38 healthy controls. We used T2‐relaxation with combined GRadient And Spin Echoes (GRASE) to measure myelin water fraction (MWF‐G), multi‐component driven equilibrium single pulse observation of T Both approaches provide WM 1 and T2 relaxation times, and by measuring the relative signal contribution from each component, a measure such as the myelin water fraction (MWF) can be obtained  is also found in the literature as a quantitative biomarker for pathology  sequence  indicate that two methods have similar tissue contrast, an important precondition. However, the level of consistency between quantities from the different methods is unclear.The common feature of most of these validation studies is that they have shown that the quantitative image 2 relaxation, mcDESPOT, MTR, and qT1. The techniques were compared against each other to determine which technique best separates tissue between MS patients and healthy controls. Finally, we investigated the relative sensitivity of each of these techniques to lesional WM in individual patients with MS.In this study, we investigated the cross‐subject similarity between four prominent quantitative MRI techniques that are associated with WM content: Multi‐echo T22.1The study cohort consisted of 56 patients with relapsing–remitting MS  recruited as part of a clinical trial of ocrelizumab versus interferon beta 1a   were performed. A 3D‐T1‐weighted gradient echo scan  was also acquired. For lesion identification, a proton density weighted (PDw) , a T2‐weighted , and a FLAIR  sequence were collected. Table All MR imaging was performed on a Philips 3T Achieva scanner  using an eight‐channel head RF array coil. For localization, a true midline sagittal scan  and a quick T2.32.3.12 relaxation was measured using a GRASE sequence   and balanced steady‐state free procession (bSSFP) acquisitions across a range of flip angles (α) as well as an inversion‐recovery‐prepared SPGR (IR‐SPGR) scan for correction of flip angle inhomogeneity Deoni, . A commos Deoni, .2.3.32, FOV = 230 × 192 × 100 mm3, α = 18°, with and without an off‐resonance RF pulse centered 1.1 kHz below the water frequency, sinc‐gaussian envelope of duration = 15 ms, bandwidth = 190 Hz, amplitude = 2.3 × 10−6 T).The magnetization transfer imaging acquisition consisted of a 3D gradient echo sequence  and implemented using the buildtemplateparallel.sh script . A final nonlinear transformation was calculated from this template space to MNI space, also using the ANTs package.To provide a representative common space for the datasets collected here, a study‐specific template was constructed, from the high flip angle (18°) T1‐weighted SPGR images from the mcDESPOT sequence using rigid registration  were registered and resampled into MNI space using nearest neighbor interpolation. The registration pipeline is outlined in Figure Within‐subject, MTR (image without the saturation pulse), MWF‐G (first echo GRASE image), and PDw images were registered to the high flip‐angle T2.62w, PDw, and FLAIR for each subject by an experienced radiologist, placing one or more seed points where a lesion was identified. Segmentation was then performed automatically using the provided seed points according to the method described by Tam, Traboulsee, Riddehough, Sheikhzadeh, and Li . Three masks were created, one for gray matter, one for WM, and one encompassing subcortical structures  map were created for each modality from the healthy control group. Spearman correlation maps between the different quantitative values at every voxel were calculated for the whole brain using 3dTcorrelate, part of the AFNI package . All analyses comparing methods or including the patient group were performed in standard MNI space and only in areas of the brain with coverage in all four modalities.Sample mean and 3.1https://neurovault.org/collections/4709/) and at https://www.msmri.com/.To assess the similarity between methods at capturing individual differences, voxelwise Spearman rank correlations between parameters are demonstrated in Figure n = 38) and the subset of patients for whom all four modalities were available (n = 24). The correlation coefficients (ρ) are summarized in Table 1  and MWF‐G and MWF‐D . In lesional tissue, for the 24 patients for whom all quantitative modalities were available, qMRI estimates covaried between all pairs of methods.Correlations across tissue classes  are reported in Table 3.2n = 24 for MTR, and n = 56 for the other methods). For all methods, significant differences between MS and controls were detectable between mean quantitative values in WM regions . Notably, these summary values for each tissue type explicitly excluded tissue identified as lesional and peri‐lesional on clinical inspection. Figure 1 values, than the control average in lesional tissue for almost every individual patient. Similarly, Figure N = 38 for all metrics, while for MS patients N = 56 for MWF‐G, MWF‐D, and qT1; and N = 24 for MTR  in the patient and control groups Figure a, indica3.3Χ2 = 32.350, df = 3, p < 0.001). Post hoc Wilcoxon signed rank tests (for paired data) indicated that MWF‐G had a significantly lower performance than all other methods (p < 0.001) and MWF‐D performed significantly better than qT1 (p = 0.017). MWF‐D had significantly higher AUC compared to both qT1 (p = 0.014) and MTR (p = 0.024). There was no significant difference between qT1 and MTR (p = 0.92). See Figure 1, and MWF‐G were only slightly  different  and at https://www.msmri.com/. These summary statistics were calculated in MNI space.In place of estimating statistical power for a limited amount of study designs/effect sizes, we include group average mean and p Figure  and prov41 observed in the present study have been demonstrated previously by  showed strong relationships throughout the brain, though these relationships varied in different tissue types. The measures were all sensitive to differences in MS tissue compared to healthy controls, though to varying degrees, and had similar sensitivity profiles to manually identified MS lesions, with MWF‐D being the most accurate. The strong associations between MWF‐D and qT1 would break down as the MWF maps will approach zero. Correlation coefficients between MWF‐D and MWF‐G indicated a moderate correlation in most tissue types. In fact, after MWF‐D and qT1, MWF‐D and MWF‐G were the pairs of measures significantly correlated across the most tissue classes. We observed weak, nonsignificant correlations in most tissue classes between MWF‐D and MTR. Similar to all other inter‐method comparisons, the correlation was significant in lesional tissue, indicating that although the methods may be measuring different quantities in healthy tissue, they are similarly sensitive in MS lesions.However, in the MS brain specifically, the combination of increased water content, demyelination, and tissue iron deposition may disrupt this relationship. In the extreme case of completely demyelinated tissue, the inverse relationship between myelin and T1. Previous results have demonstrated a lack of correlation between MWF‐G and MTR in nonlesional WM , and maybe the Gareau investigation (n = 24 as well), there may simply have been insufficient samples to detect weak relationships. Several in vivo studies suggest that MTR is most strongly influenced by water content  in WM, but similar to previous studies, we find a strong correlation in lesions, most likely due to an increase in water content.There is a more established history in quantifying the similarity between MWF‐G, MTR, and qTIn addition to characteristic focal lesions, there are diffuse abnormalities in the MS brain and notably: differences in brain volume . To improve on this, many studies have combined multiple image modalities, both qualitative and quantitative, to produce more accurate lesion segmentations , this could falsely induce or occlude associations between methods. Although spatial smoothing may help, matching acquisition dimensions can better address the problem. In addition, the fact that the mcDESPOT acquisition was used for nonlinear registration means that errors in registration are less likely for qT1 and MWF‐D compared to the other two methods where an additional rigid registration is needed and error may be introduced just to MTR and MWF‐G due to the compound registration approach. In all cases however, multiple interpolation steps were avoided by combining the calculated transformations and interpolating from native to MNI space in a single step.This brings up another methodological limitation when comparing the different methods. Limitations on matching image resolution across acquisitions mean that voxels can have different aspects of partial volume to each other. Therefore, while MWF‐D and qTSD of the four quantitative MRI techniques (https://neurovault.org/collections/4709/).In summary, we demonstrate that individual differences of WM tissue content are not consistent between WM quantification techniques in the same individuals. Although important in itself, this is especially important as these techniques are being used more and more in research, referred to interchangeably, though they are obviously quite different. These techniques are also being increasingly used in clinical trials of WM disorders and, where pathological tissue is concerned, we show reassuringly good correspondence between the techniques. However, depending on the exact purpose of a study, one technique may be preferred over another. For studies that require the greatest confidence in the interpretation of results as changes specific to myelin, MWF‐G has undergone the greatest degree of validation and is thought to provide the greatest specificity. If sensitivity to differences in tissue types  is more important than the strictest specificity, MWF‐D and qT1 showed the greatest separation. Consequently, these techniques are the most likely to be sensitive to change over time. If acquisition or analysis times are severely limited, MTR or qT1 provide the most practical tools. To enable bespoke power analyses for future trials and studies, we provide voxelwise estimates of the sample mean and


PDAC bone metastases represent a clinical challenge characterized by multifaceted biological entity. He did not report trauma or comorbidities for osteoarticular pathologies in his past medical history. Drugs prescribed for pain control did not relieve symptoms. X‐ray examination showed mild lumbar right‐convex scoliosis and signs of osteophytic spondylosis. Vertebral discomfort and aggravating pain at the level of D11 and D12 vertebrae prompted a neurological assessment which revealed slight inferior limb numbness and weakness, mainly in the left leg in combination with hypoesthesia and dysesthesia in the same region; perineal reflexes were present. Computerized tomography images of vertebral column revealed bone rarefaction with D11 vertebral fracture and spinal cord compression  and nab‐paclitaxel reduced fibrosis, peritoneal dissemination, angiogenesis, and cell proliferation.P = .025), with a 6‐month survival rate of 33%. Moreover, the same authors demonstrated that radiotherapy of 1 × 8 Gy appeared to be not inferior to multi‐fraction radiation schedules considering the post‐treatment motor function in patients with vertebral involvement in PDAC.Scanty data are available in the literature concerning the clinical outcome of PDAC patients with bone metastases. Borad et al reported a series of seven patients who developed skeletal metastases between 2 and 17.3 months (median: 5.5 months) after the onset of the disease. Survival from the evidence of skeletal metastases ranged between 0.3 and 9 months. Most of these patients (71.4%) received bisphosphonates in combination with systemic chemotherapy.Survival of our patient was lower than the median overall survival of a metastatic PDAC patient treated with first‐line gemcitabine and nab‐paclitaxel. In previous reports, we described a relationship between bone metastases and poorer prognosis in patients affected by gastric cancer and hepatocellular carcinoma.Conclusively, we hypothesize that bone metastatization could represent the phenotypic expression of the underlying molecular PDAC subgroups characterized by unfavorable outcome. This patient's subgroup might be characterized by peculiar prognostic feature, being potentially candidate to specific targeted therapies.The authors have no conflict of interests.AA, OB, AGS: Conceptualization, AC, AA, AN and AC: methodology, MMGP, AA, AN, AC and AGS: formal analysis, AA, OB and AGS: investigation, AA, MMGP and AN: data curation, AA, OB and AGS: writing—original draft preparation, AA and OB: writing—review and editing, AA and OB: resources, OB and AGS: supervision, AGS: funding acquisition.


Similar patterns were found in waxcap basidiocarps from diverse habitats across four continents. Additional data from 14C analysis of basidiocarps and 13C pulse label experiments suggest that these fungi are not saprotrophs but rather biotrophic endophytes and possibly mycorrhizal. The consistently high but variable δ15N values (10–20‰) of basidiocarps further indicate that N acquisition or processing differ from other fungi; we suggest that N may be derived from acquisition of N via soil fauna high in the food chain.Several lines of evidence suggest that the agaricoid, non‐ectomycorrhizal members of the family Hygrophoraceae (waxcaps) are biotrophic with unusual nitrogen nutrition. However, methods for the axenic culture and lab‐based study of these organisms remain to be developed, so our current knowledge is limited to field‐based investigations. Addition of nitrogen, lime or organophosphate pesticide at an experimental field site (Sourhope) suppressed fruiting of waxcap basidiocarps. Furthermore, stable isotope natural abundance in basidiocarps were unusually high in  However, their resistance to axenic culture has hindered study of their biology, so they are often (by default) considered as saprotrophs. Here we combine field surveys assessing the effects of agricultural manipulations on fruiting with isotopic  analyses to show that they are biotrophs, potentially forming mycorrhizal associations with host plants. Whilst the 13C and 15N patterns of their basidiocarps resemble ectomycorrhizal fungi more than saprotrophs, their consistently high δ15N values also indicate an unusual mode of nitrogen nutrition, for which we suggest some possible mechanisms.This manuscript presents the results of experiments to elucidate the nutritional biology of soil‐dwelling Hygrophoraceae (waxcaps). This major group of agaricoid fungi is primarily known from undisturbed grassland habitats where they are dominant components of the soil fungal community, more abundant than arbuscular mycorrhizal fungi  and cyanobacteria (Dictyonema spp.) to ectomycorrhizal (ECM) taxa (Hygrophorus spp.)  in undisturbed grasslands. Waxcap basidiocarps are much rarer or absent in grasslands subject to agricultural intensification so these fungi have suffered large‐scale habitat loss, especially in lowland areas. Recent investigation of grassland fungi using DNA metabarcoding has shown that Hygrophoraceae are amongst the most abundant fungi in undisturbed grassland soils , most likely by default (i.e. lack of published evidence to the contrary). However, several lines of evidence now suggest that they are biotrophic, similar to their lichenised and ectomycorrhizal relatives in family Hygrophoraceae, amongst which they are interspersed , where endophytism acts as a symbiotic “waiting room”  have been used to examine the nutritional strategies, trophic position and feeding behaviours of diverse organisms , these exhibit 15N enrichment rather than depletion  were found on the Sourhope plots with six additional species found outside plots but within the fenced 1 ha experimental area  and Hygrocybe ceracea (12%).The negative effects of synthetic fertilizers on the fruiting of waxcaps have been anecdotally reported  biocide were applied at standard agricultural levels, lime was applied at five times the usual rate, leading to a substantial increase in pH on limed plots, from 4.5 in 1999 to > 7 in 2003 , significantly lower in 15N than basidiocarps, whereas δ13C values resembled soil and vegetation values  during basidiocarp field surveys. The δe to 0‰ − to +2‰, ues Fig. . Soils ath Figs. , followi15N values, ranging from 11.7 to 20.6‰ .During field surveys at Sourhope and other sites, all basidiocarps (waxcap and any others) were mapped. Representative samples from each plot and each survey visit were collected and dried, and a subset of these were sent for isotopic analysis. Basidiocarp caps collected from control plots at Sourhope had high δ15N did not differ significantly among different age classes  but δ2 via the Haber‐Bosch process has a δ15N value close to zero , England , Bavaria  and Liguria  but samples from Iceland were less highly enriched . Excluding Iceland, there was consistent 15N enrichment, with all but one  of the 302 basidiocarps sampled having a δ15N signature greater than 10‰ (range 10.9–20.9‰). There was neither significant difference in δ15N values for samples from the five genera and 21 species within grassland Hygrophoraceae, nor did grassland type significantly affect δ15N (lowland vs. upland).Similar patterns for isotopic values of waxcap basidiocarps from Sourhope were found in further analysis of samples from upland and lowland grassland sites in Wales, England, Germany, Iceland and Italy Table , includi15N  to grassland samples. However, two samples of C. lacmus from a stand of pure heather on Lundy Island were slightly lower in δ15N (ca. 9.5‰), whilst two samples of H. cantharellus from a boggy upland site vegetated with Sphagnum moss had very low δ15N values (0.5 and 1.8‰)  than other Hygrophoraceae. This was the case here for three additional UK and New Zealand Hygrophorus specimens.In Europe, waxcaps are only rarely encountered beyond grassland habitats but four samples from UK woodlands were similarly enriched in 15N ;subtropical laurel (laurisilva) montane forest in the Canary Islands , Cyathea spp. , Vitex lucens (Lamiaceae), Dysoxylum spectabile (Meliaceae) or Leptospermum scoparium (Myrtaceae), with some remaining canopy trees . , mostly secondary forest dominated by  Barton, ;primary lowland moist evergreen forest in Amazonian Ecuador (Cuyabeno Forest Reserve) ; La Palma: ; Ecuador ; Gabon: . However, in New Zealand and La Palma, both areas where the vegetation is dominated by non‐ectomycorrhizal Lauraceae, the range of δ15N values was particularly high and included some samples where δ15N enrichment was much lower than found in European grasslands. These lower (< 10‰) values were all found in members of genus Hygrocybe Subgenus Pseudohygrocybe, Sections Coccineae or Firmae . Samples from New Zealand varied widely in δ13C values (−23.9 to −32.1‰) and were over‐represented relative to other areas amongst the more extreme δ13C values.Waxcap basidiocarps were lower in δge) Fig. . The dat14C content of three Hygrophoraceae samples were analysed, alongside two samples of saprotrophic fungi (Agaricus campestris and Cystoderma amianthinum) which commonly co‐occur with waxcaps. Samples collected during the period 1970–1985 (from the RBG Kew Fungarium) were selected, as the steeper gradient then of the atmospheric 14C curve allowed more accurate dating. The calibrated midpoint ages for A. campestris and C. amianthinum were 5.0 and 4.4 years, suggesting that basidiocarp carbon was derived from organic matter formed several years earlier , all from the roots of non‐fagaceous (non‐ECM) plants. Thus waxcaps appear to avoid ectomycorrhizal plants  values for European waxcaps were for two H. cantharellus samples in Sphagnum. Several of the Hygrocybe samples analysed by Seitzman et al. . H. cantharellus is found with Sphagnum in New Zealand (http://www.kaimaibush.co.nz/Fungi/Hygrocybe1.html) and several Hygrocybe spp. from this area had unusually low δ15N, however, precise substrate details are not available for our samples. Thus the association of certain Hygrocybe spp. with Sphagnum, and the low δ15N values for these at diverse locations, suggests a biotrophic interaction atypical of those more commonly found amongst waxcaps. This may relate to the important role of cyanobacteria in the nitrogen nutrition of Sphagnum  in Welsh grasslands  than are those of ECM , as well as saprotrophs from other, mostly woodland, habitats , consistent with the preferential loss of 12C as CO2 during microbial catabolism of organic matter  were, however, similar to vegetation and slightly depleted in 13C relative to soil by ca. 2‰. If waxcaps are saprotrophic, it is difficult to explain why they are depleted in 13C relative to soil, and distinct from known saprotrophs. Thus the most parsimonious explanation for the 13C natural abundance patterns reported here is that that they derive C directly from plant photosynthate.We did not undertake extensive analyses of associated soil and vegetation but at Sourhope δ14CO2 during the 1970–1980s to estimate accurately the date at which the carbon present in fungarium basidiocarps had been fixed by photosynthesis. The 14C age of the waxcap samples analysed was 0–2 years, whereas for two saprotrophic grassland fungi, the 14C age was 4–5 years. These 14C dates for waxcaps are consistent with the use of recently fixed C such as photosynthate, and not consistent with the use of soil organic matter which contains C fixed many years earlier  but this respiration could also have been by mycelia of waxcaps, a possibility supported by DNA metabarcoding studies which consistently find AMF abundance (< 5%) in undisturbed grasslands to be lower than that of waxcaps and other fungal groups . The most heavily labelled PLFA was 18:2ω6,9 which is the dominant PLFA in basidiomycetes  and present at only low abundance (< 2%) in AMF  associated with mycoheterotrophic orchids , however, values above 10‰ are unusual  at the mycorrhizal interface  which mediates release of N‐acetylglucosamine monomers but it lacks the requisite genes for further breakdown [N‐acetylglucosamine‐6‐phosphate deacetylase (EC3.5.1.25) and glucosamine‐6‐phosphate deaminase (EC3.5.99.6)] which are present in the genomes of most saprotrophic fungi hitherto studied (http://www.genome.jp/kegg). This suggests, for this species at least, that once incorporated into chitin, this N is not released back to the protein/inorganic pool.Transamination from glutamine is also involved in chitin synthesis . Whether this is a feature of all mycorrhizal association is unclear as both  form only limited mycelial networks , , similar to that found for this species at a global scale. Even for basidiocarps forming part of the same fairy ring there was 3–4‰ variation , whilst Neilson et al. (15N (8–10‰) of all the earthworm species at several of the grassland sites they studied. Similarly, Schmidt et al.  seasonal variation that was recorded. This corresponds to 1.75–3.5 g N.m−2 (assuming 83% water content and 10% N content of dw), ca. 1% of the total N pool [for Sourhope soils  =240 g N.m−2]. We further estimated the total N content in waxcap biomass to be ca. 3 g.m−2 ; lime ; biocide ; Nitrogen/Lime  : Control , nitrogen fertilizer [N; granular NHIsotopic analyses were conducted on cap (pileus) tissues of dried basidiocarps. Samples observed during field surveys were picked, dried within 24 h of collection in a ventilated drying cabinet at <40°C and stored desiccated at the Aberystwyth University Fungarium (code ABS). Identification of samples was based on macroscopic and microscopic examination according to Boertmann . Subsamphttp://www.bayceer.uni-bayreuth.de/ibg/en/ausstattung/geraet/geraet_detail.php?id_obj=65905). Values were referenced against atmospheric nitrogen and V‐PDB limestone standards.Isotope analyses were conducted by continuous flow‐isotope ratio mass spectrometry (CF‐IRMS), using an automated N/C analysis‐mass spectrometry (ANCA‐MS) system  at Lancaster, North Wyke, by Conflo III Interface. Recent samples from Italy, Liguria, La Palma and northern Bavaria were analysed at the Bayreuth University (for details refer to 14C) levels were elevated, three Hygrophoraceae samples were chosen from the Mycological Herbarium at the Royal Botanic Gardens (RBG), Kew. Samples were taken from grassland sites but avoided urban areas, where the 14C‐dead CO2 from localized fossil fuel burning could cause distortion of data. The samples were analysed to quantify 14C content. Basidiocarp cap segments were subjected to a weak acid wash (0.5 M HCl) to remove potential contaminants. The samples were then combusted in sealed quartz tubes and sample CO2 cryogenically recovered, graphitised  at the Scottish Universities Environmental Research Centre, East Kilbride, UK. Following convention, 14C results were normalized to a δ13C of −25‰, and expressed as percentage modern (ratio of sample 14C content relative to the oxalic acid international standard; (Stuiver and Polach, 14C content over the last 60 years. This calibration was performed using ‘Calibomb’ software (Reimer et al., 14CO2 dataset (Levin et al., To obtain basidiocarp samples corresponding to a period when atmospheric radiocarbon (Suppdata 1. Details of Sourhope surveysSuppdata 2. δ13C and δ15N values of soils and vegetation at Amorbach and Sourhope.Suppdata 3. Sampling locations for basidiocarp isotope analysesSuppdata 4. δ15N / δ13C in basidiocarps of different ages between cap vs. stipe tissues.Suppdata 5. Images of C. lacmus and H. cantharellus growing in heather/mossSuppdata 6. Spatial distribution of C. pratensis and C. virgineus basidiocarps at SourhopeSuppdata 7. Data from the seven waxcap Basidiocarps close to pulse sitesSuppdata 8. Summary of Hobbie and Agerer theoretical pattern of N isotope fractionation in mycorrhizal systemsSuppdata 9. δ15N values in soil invertebrates from published studiesSuppdata 10. Raw data and references for published studies relating to Fig. 3Click here for additional data file.


Brainstem raphe (BR) hypoechogenicity in transcranial sonography (TCS) has been depicted in patients with major depression (MD) and in depressed patients with different neurodegenerative diseases. But, up to date, the association of BR alterations in TCS with depression in migraineurs has never been reported. This study was to investigate the possible role of BR examination via TCS in migraineurs with depression.Forty two migraine without aura (MwoA) patients and 40 healthy controls were recruited. Echogenicity of lentiform nuclei (LN), caudate nuclei (CN), substantia nigra (SN) and brainstem raphe (BR) and width of the frontal horns of the lateral ventricles and the third ventricle were assessed with TCS. The diagnosis of depression was based on the criteria of the Diagnostic and Statistical Manual of Mental Disorders IV (DSM –IV), and the severity of depression was measured by Hamilton Rating Scale for Depression (HAM-D) and Hospital Anxiety and Depression Scale depression subscale (HADS-D).p = 0.955), width of third ventricle (p = 0.129) as well as in the echogenicity of SN (p = 0.942), CN (p = 0.053), LN (p = 0.052) and BR (p = 0.677). Here, it seems that more migraineurs were detected with increased echogenecity of CN and LN compared with controls  though they had no statistical significance. Patients with hypoechogenic BR had significantly higher HAM-D and HADS-D scores than those with normal BR signal (p = 0.000 for both HAM-D and HADS-D), and most (83.33%) migraineurs with depression exhibited hypoechogenic raphe but none (0.00%) of the migraineurs without depression exhibited hypoechogenic raphe (p = 0.000).There were no significant differences between migraineurs and controls in the width of frontal horn of the lateral ventricle (TCS signal alteration of BR can be a biomarker for depression in migraine but it is not associated with migraine headache itself. LN and CN alterations in TCS may reflect a potential role of them in the pathogenesis of migraine, which needs to be further elucidated. Transcranial sonography (TCS) is a reliable and non-invasive neuroimaging technique for detecting small deep brain parenchyma such as basal ganglia (BG), substantia nigra (SN) and brainstem raphe (BR) . The hypThe comorbid relationship between migraine and depressive disorder are well-known. The risk of comorbid depressive disorder in migraineurs is over 2.5 times higher than in non-migraineurs , 10, visIn order to investigate the possible role of BR in migraine with depression, migraine without aura (MwoA) patients with or without depression will be included in this study. Possible alterations of BR echogenicity in TCS in migraineurs will be studied in comparison with the healthy control. And possible difference in BR echogenicity between migraineurs with and without depression will be studied. On the other hand, possible alterations of the width of frontal horn of the lateral ventricle and the third ventricle as well as of the echogenicity of substantia nigra(SN), caudate nuclei (CN) and lentiform nuclei (LN) will also be studied, as alterations of these deep brain structures have been identified either in migraine –19 or inA total of 42 patients fulfilling the International Headache Society (IHS) classification criteria for MwoA  were proTCS examination was conducted by Dr. Xue-Gong Shi, an experienced sonographer who has been engaged in ultrasound for 26 years and he was blinded to clinical scores of the enrolled participants in this study. A phased-array ultrasound system equipped with a 1.6–2.5 MHz transducer  was employed for the examination. A dynamic range of 45–55 dB and penetration depth of 14–16 cm through the transtemporal bone window were used , 24. For2 is considered normal, larger than 0.20 cm2 is rated as hyperechogenicity [TCS examination was carried out on standardized axial imaging planes as recommended for deep brain parenchyma detection . At the genicity   if it is normally distributed, or else as median . For comparison of categorical variables, the chi-square test was performed. For comparison of non-normally distributed data, the Mann-Whitney U test was used. And for comparison of means, the t-test for independent samples was used. A p = 0.001) and HADS-D score  . The width of the third ventricle (> 7 mm) was found abnormal in one migraine patients (4.8%) and normal in the rest migraineurs and controls. Mean width of third ventricle is 4.13 ± 1.26 mm and 3.77 ± 0.81 mm in migraineurs and controls respectively (p = 0.129). Hyperechogenic SN was present in 4 migraineurs (9.5%) and 4 controls (10.0%). Mean area of SN is 0.16 ± 0.03 cm2 in migraineurs and 0.15 ± 0.04 cm2 in controls (p = 0.308). CN hyperechogenicity was found in 14 migraineurs (33.3%) and 6 controls (15.0%). And hyperechogenicity of LN was found in 8 migraineurs (19.0%) and 2 controls (5.0%). Hypoechogenicity of BR was present in 10 patients (23.8%) and 8 controls (20.0%), and BR echogenicity was undetectable in 1 out of the 10 patients with BR hypoechogenicity but in none of the healthy controls. There were no significant difference between migraineurs and controls in the width of frontal horn of the lateral ventricle (p = 0.955), width of third ventricle (p = 0.129) as well as the echogenicity of SN (p = 0.942), CN (p = 0.053), LN (p = 0.052) and BR (p = 0.677)  in all migraineurs and controls, mean width was 4.62 ± 0.98 mm in migraineurs and 4.63 ± 1.30 mm in controls (p = 0.000). Mean HADS-D score was 12.6 ± 3.8 (range 7–18)in patients with hypoechogenic BR, and 4.6 ± 2.7 (range 0–11)in patients with normal BR (p = 0.000). Additionally, one patient with undetectable BR echogenicity had obviously depressive symptoms with HAM-D score at 22 and HADS-D score at 18. Nevertheless, we found no significant differences between two groups in gender, age, disease duration and migraine headache attack frequency (Table p = 0.000).Our study showed that patients with hypoechogenic BR had higher HAM-D and HADS-D scores than those with normal BR. Mean HAM-D score was 18.6 ± 4.6 (range 11–24)in patients with hypoechogenic BR, and 6.1 ± 4.6 (range 0–14)in patients with normal BR (To our knowledge, this is the first study to correlate alterations of BR echogenicity with depression in migraine patients. The main TCS finding is that migraineurs exhibiting a pathologic echogenicity of BR tend to have more depressive symptoms than those patients with normal raphe echogenicity. In addition, it could be shown that the overwhelming majority of migraineurs with depression exhibited hypoechogenic raphe, while none of the migraineurs without depression exhibited hypoechogenic raphe.Some MRI studies have shown structural and functional alterations of BG in migraine patients , 27, 28,Multiple genetic and biochemical studies have demonstrated that serotonergic system is involved in migraine pathophysiology . And manAmounts of studies have shown that raphe hypoechogenicity existed in MDD patients and in depressed patients with different neurodegenerative disease such as PD, Wilson’s disease, Dystonia and Huntington disease . In our We acknowledge that the migraine headache frequency was relatively low and its range was narrow in our studying subjects, which may affect the analysis of the association of migraine itself with the echogenecity of BR as well as with that of CN and LN. In fact, very subtle structural alterations with corresponding anatomical locations such as SN, CN, LN and BR can exist, which may be overlooked when examining at the sonographic level. The aim of this study was not to investigate the possible role of this deep brain parenchyma in the pathogenesis of migraine but to determine whether the alteration of BR echogenecity could be a biomarker of depression in migraine patients. It should also be acknowledged that no double control was utilized and only one sonographer conducted the ultrasound examination in this study.In TCS level, there was no significant difference between migraine patients and normal controls in deep brain parenchyma including brain ventricles and brainstem nuclei, but it seems that more migraineurs were detected with increased echogenecity of CN and LN compared with controls. Migraine patients with BR hypoechogenicity had significantly higher HAM-D and HADS-D scores than those with normal BR echogenicity, and overwhelming majority of migraineurs with depression exhibited hypoechogenic raphe but none of the migraineurs without depression exhibited hypoechogenic raphe. This indicates that TCS signal alteration of BR can be a biomarker for depression in migraine but not be associated with migraine headache itself. LN and CN alterations in TCS may reflect a potential role of them in the pathogenesis of migraine, which needs to be further elucidated.


LEP gene with the risk of cancer have yielded inconsistent results. In this study, we have carried out a case‐control study [1063 esophagogastric junction adenocarcinoma (EGJA) cases and 1677 controls] in a Chinese population. Furthermore, we carried out a pooled‐analysis of 13 studies involving 8059 cancer patients and 11 930 controls to assess whether the LEP G19A locus was associated with overall cancer susceptibility. Odds ratios (ORs) with the corresponding 95% confidence intervals (CIs) were harnessed to evaluate the potential association. In our case‐control study, we found an association between the carriers of LEP 19A allele and EGJA risk. In addition, the results of meta‐analysis also suggested significant associations with cancer risk . Upon conducting a stratified analysis, we found that LEP 19A allele might decrease the susceptibility of non‐Hodgkin lymphoma (NHL) and colorectal cancer (CRC). In a stratified‐by‐ethnicity analysis, significant associations were also found in Asians, Caucasians, and mixed populations. We can conclude that the LEP G19A polymorphism constitutes a decreased risk of cancer.Accumulating evidence has suggested that leptin (LEP) is very important for the development of cancer. Recently, a number of case‐control studies about the relationship of the rs2167270 G > A (G19A) variants in the  LEP, a 16 kDa glycol‐protein, is predominantly made >95%) by fat cells.5% by fatLEP rs2167270 G > A (G19A) locus is correlated with LEP levels and may also give a fascinating insight into the potential correlations with the development of cancer.LEP 19A allele might have a lower tendency for cancer risk.LEP G19A polymorphism and cancer risk have been performed in Asians with relatively large samples.It is found that the LEP G19A polymorphism with the susceptibility of developing esophagogastric junction adenocarcinoma (EGJA). And then, we performed a meta‐analysis to estimate the relationship of this polymorphism with overall cancer risk.In this study, to acquire an understanding of the relationship between LEP polymorphism and risk of cancer, we first studied 22.1LEP G19A polymorphism was detected by SNPscan genotyping assay  according to conditions described by Chen et al.A total of 1063 unrelated EGJA cases were diagnosed and selected at Fujian Medical University Union Hospital, Fujian Medical University Cancer Hospital and Affiliated People’s Hospital of Jiangsu University. In addition, 1677 noncancer subjects were included in a control group. Both groups belonged to the Chinese Han populations from eastern China. The patients included 759 males and 304 females; the average age was 64.19 ± 8.63 years. Of them, 625 patients had lymph node metastases. There were 305 stage I/II and 758 stage III/IV EGJA patients included in the case group.2.2We performed an extensive literature search in PubMed and EMBASE databases, covering all medical publications until 24 August 2018, with the following key words: LEP gene (e.g.: ‘LEP’ or ‘leptin’), cancer , and polymorphism . In addition, we also carried out a manual search of the listed references of the included publications and related reviews.LEP G19A polymorphism with risk of cancer; (c) genotypes data listing in the publications. The major exclusion criteria of studies were as follows: (a) reviews; (b) duplicated reports; (c) not case‐control study designs; (c) lack of data for genotype frequencies.The criteria of literature selection were as follows: (a) investigation designed as a case–control study; (b) focusing on the association of Two authors (J. Yang and Z. Zhong) extracted data from the included publications independently. The following information was collected: (a) first author; (b) publication year; (c) number of cases and controls; (d) country; (e) ethnicity; (f) source of controls; (g) cancer type; (h) genotyping method; and (I) genotype frequency. Ethnicities were defined as mixed, Asians, and Caucasians. For the source of controls, the publications were categorized as hospital‐based and population‐based studies.https://ihg.gsf.de/cgi-bin/hw/hwa1.pl). The strength of the correlation between LEP G19A locus and cancer risk was determined by calculating crude odds ratios (ORs) with their 95% confidence intervals (95% CIs). The following four models were calculated: homozygote comparison (AA vs GG), dominant model (AA/GA vs GG), recessive model (AA vs GG/GA), and allele model (A vs G). If I2 > 50% or P < 0.1, it suggested that there was significant heterogeneity. Considering the heterogeneity among the included studies, a different model was used to pool the data. When no significant heterogeneity was identified, the Mantel‐Haenszel method (fixed effects model) was usedP < 0.1 was defined as representative of significant bias. The Newcastle‐Ottawa Quality Assessment Scale was used to assess the quality of the enrolled literatures. If scores ≥ 6 stars, the publication was considered as related high‐quality. In this study, all P values for statistics were calculated with two‐sided. STATA 12.0 software  was used to analyze the data.In this study, we analyzed Hardy‐Weinberg equilibrium (HWE) using a goodness‐of‐fit test using an online software  were included in this case‐control study. Table LEP G19A polymorphism. In the analysis of LEP G19A polymorphism, differences in the distribution of LEP G19A genotypes between EGJA patients and controls were found . The results of multivariate linear regression analysis also showed that LEP G19A polymorphism was correlated with a decreased risk of EGJA .Table 3.2We have summarized the meta‐analysis process in Figure LEP 5′‐UTR region with a decreased risk of overall cancer in four genetic models .As demonstrated in Table LEP 19 A allele might have a lower susceptibility of NHL in three models . In addition, we also found that the G19A polymorphism in LEP gene was correlated with a decreased susceptibility of CRC in homozygote comparison  and recessive model .When an analysis stratified by cancer type was conducted, we found that individuals carrying P = 0.005), Caucasians for three models , and mixed population .In an analysis stratified by ethnicities, significant associations were found also in Asians , gene‐environment interaction could not be carried out. Third, in this study, only LEP 5′‐UTR region is associated with a decreased risk of EGJA. In addition, the subsequent meta‐analysis also indicates that this SNP decreases the risk of overall cancer. To confirm or refute our findings, large scale case‐control studies are needed.In conclusion, it is highlighted that the G19A polymorphism in the The authors declare that they have no conflicts of interest.


Bipolar disorder is associated with heightened and persistent positive emotion (Gruber in Curr Dir Psychol Sci 20:217–221, The current study adopted eye-tracking methodology as a continuous measure of sustained overt attention to monitor gaze preferences during passive viewing of positive, negative, and neutral standardized photo stimuli among remitted bipolar adults and healthy controls. Percentage fixation durations were recorded for predetermined areas of interest across the entire image presentation, and exploratory analyses were conducted to examine early versus late temporal phases of image processing.Results suggest that the bipolar and healthy control groups did not differ in patterns of attention bias.Findings provide insight into apparently intact attention processing despite disrupted emotional responding in bipolar disorder. Bipolar disorder (BD) is a severe and chronic psychiatric condition associated with functional impairment and disability . The other non-mutually exclusive hypothesis predicted that individuals with BD would demonstrate specific attentional biases away from negative emotional stimuli (i.e. negative attenuation).’The present investigation aimed to explore attentional biases toward emotionally relevant stimuli in BD by employing continuous eye-tracking during individually presented images. By presenting images one at a time, the current study constrained participants’ potential attentional biases to affectively salient vs. non-salient areas, mimicking visual field processing in a more ecologically valid way. Using this methodology, individual and age-group differences in attentional biases have been found within non-clinical populations , and outpatient mental health referrals. Participant sample size was based on common standards in research with severe psychiatric samples at the time of recruitment  and included 35 individuals diagnosed with BD type I, currently remitted, and a healthy control (CTL) group comprising 32 individuals who did not meet current or lifetime criteria for any DSM-IV-TR Axis I disorder  and 28 negative  images , but this is typical for research utilizing neutral IAPS stimuli , followed by a buffer slide with a fixation cross (0.5 s) to realign gaze to the center of the screen.An Applied Science Laboratories eye tracker, Model 504  recorded the movements and position of participants’ left eye sixty times per second with a camera and a non-invasive beam of infrared light. The visual fixations were defined as those series of gazes in which an individual stayed within 1° visual angle for 100 ms or longer  to understand overall attentional biases for different image valences. Second, we examined group differences in early (0.0–2.5 s) and late (2.51–5.0 s) phases of image presentation separately  to understand group differences in attentional focus towards images in the later phase of image viewing after accounting for fixation duration in the early phase. This analysis was exploratory in nature, and implemented post hoc after discovering that visual attention in later stages (i.e. 10–15 s compared to 1–10 s) of visual attention to positive stimuli has been positively associated with trait levels of happiness within non-clinical subjects , and groups did not differ in percentage of trackable data  = 0.92, p = 0.34, η2 = 0.001).Following convention in previous eye-tracking studies . Not surprisingly, the BD group scored lower on global functioning than the CTL group. Although all groups scored below YMRS (≤ 7) and IDS-C (≤ 11) cutoffs, BD participants scored higher than CTL participants on the IDS-C and YMRS, and thus symptoms were controlled for in the main analyses. The groups did not differ on the baseline cognitive measures (p > 0.05). There were no main effects for order (ps > 0.13) or gender (ps > 0.12) for all main variables. Finally, BD participants were currently remitted for an average of 27.57 months (SD = 31.99).As seen in Table SD = 9.75), recruited via Amazon Mechanical Turk . AOIs within positive images were rated as significantly more pleasant  and arousing  than the rest of image area , t(99) = 8.85, p < 0.001 for valence and t(99) = 6.14, p < 0.001 for arousal. Similarly, AOIs within negative images  were rated as significantly less pleasant  and more arousing  than the rest of the image areas , t(99) = − 11.47, p < 0.001 for valence and t(99) = 8.234, p < 0.001 for arousal.To ensure the validity of our emotional stimuli 100 independent judges ; therefore, degrees of freedom were corrected using Huynh–Feldt estimates of sphericity. Results revealed a main effect of Valence, F = 54.35, p < 0.001, η2 = 0.55, with more percent time fixated on Neutral images , followed by Positive images , and Negative images , thus not supporting the positive amplification nor negative attenuation hypotheses. The Group main effect (p = 0.50) and Group x Valence interaction were not significant (p = 0.36). Pairwise comparisons for each Valence type were conducted to compare the BD versus the CTL group but were not significant, (ps > 0.21). No significant differences were found in fixation duration patterns for the entire image presentation time.In order to examine the differences in sustained attention between the BD and CTL groups we conducted a 2  × 3  repeated-measures ANCOVA for the average percent time participants fixated on emotional AOI across the entire image presentation (5.0 s), while controlling for depression and manic symptom levels (YMRS and IDS-C scores).emotional AOI separately for the early and late phases of image presentation by computing a difference score by subtracting fixation % from the late − early phase scores  = 2.95, p = 0.06, η2 = 0.06. No main effect of group (p = 0.79) or group × valence interaction effect was found, (p = 0.43). None of the between-subjects comparisons were found significant, ps > 0.34.Next, we conducted exploratory analyses to explore whether attentional profiles were sensitive to the temporal dynamics of visual attention by examining the average percent time participants fixated at es Table . There wd, p = 0.3. None oremitted BD  is a chronic psychiatric disorder that is associated with heightened and persistent positive emotion and reward sensitivity, even during periods of remission from mood episodes  of an image. These findings broaden our understanding of emotion processing in BD by assessing whether visual attention is differentially allocated within a single emotional image, rather than between several emotionally valenced images. This distinction is important, as visual attentional tasks typically present simultaneous, distinct options for gaze, which may not mirror ecologically-valid visual scenes that include both emotionally salient regions as well as less emotionally salient regions. Overall, the results suggest that remitted BD is not characterized by differential visual attention biases to affective regions of stimuli compared to non-clinical controls.Findings from the present study should be interpreted within the confines of several limitations. First, although the images used in the present study are standardized and reliable elicitors of emotions, it could be argued that the results may not be generalizable to everyday emotional experiences or visual scenes in the lives of BD patients. Thus, it will be important for future studies to assess attention biases using stimuli that are more ecologically valid , and perhaps more dynamic in nature  and personally salient . Second, the use of complex static images may have introduced variance in emotional gaze responses, which may have driven observed attentional patterns towards or away from the image (e.g., Raila et al. While the current study was not designed to investigate emotion regulation, it sheds light on an important cognitive component of emotion regulation; namely attention allocation to affective stimuli or situations Gross . Difficu


PDAC) there are only four abundantly common driver mutations , which are not currently actionable. Thus, precision therapy for PDAC must look beyond the cancer cell. In fact, PDAC is more than a collection of renegade cells, instead representing an extensive, supportive ecosystem, having developed over several years, and consisting of numerous interactions between the cancer cells, normal mesenchymal cells, immune cells, and the dense extracellular matrix. In this issue, Huang and colleagues demonstrate how elucidation of these complex relationships within the tumor microenvironment (TME) can be exploited for therapeutic intervention in PDAC. They identify in a subset of PDAC with mutations in TGFβ signaling, that a paracrine signaling axis can be abrogated to modulate the TME and improve outcomes.Cancer can be conceptualized as arising from somatic mutations resulting in a single renegade cell escaping from the constraints of multicellularity. Thus, the era of precision medicine has led to intense focus on the cancer cell to target these mutations that result in oncogenic signaling and sustain malignancy. However, in pancreatic ductal adenocarcinoma ( Despite advances in first‐line therapy, 5‐year survival has only increased from 5 to 8% over the last 30 years  in both processes. In this issue of EMBO Molecular Medicine, KrasLSL‐G12D/+; Cdkn2aflox/flox; Ptf1aCre/+) and KPC . After treating these mice with 2G8, analysis of the stromal secretome identified consistently diminished IL‐6 levels. The IL‐6 cytokine has a well‐established role in promoting PDAC growth through JAK/STAT signaling  including KIC  cell activation by RNA sequencing. In fact, vity Fig .et al, et al, However, despite abrogation of TGFβ‐induced IL‐6 production by CAFs, reduced pSTAT3 in cancer cells, and reprogramming of the immune microenvironment, 2G8 treatment resulted in decreased survival. This was not entirely unexpected as inhibition of TGFβ signaling in PDAC has previously been unsuccessful (Hezel Taken together, this extensive study from


EGFR)‐tyrosine kinase inhibitor treatment has been reported, but details of the transformation remain unclear. We report two cases with transformation to squamous cell carcinoma. The first case was a 61‐year‐old man who was an ex‐smoker with stage IV lung adenocarcinoma harbouring EGFR exon 19 insertion. He experienced squamous cell transformation after 28 months of erlotinib therapy. Next‐generation sequencing (NGS) analysis showed EGFR T790M and genomic alterations in PTEN, PDGFR, and HRAS. The second case was a 72‐year‐old man who was an ex‐smoker with stage IV lung adenocarcinoma harbouring EGFR exon 21 L858R. He experienced squamous cell transformation after nine months of erlotinib therapy. NGS analysis showed EGFR T790M and genomic alterations in PTEN, SMARCB1, TP53, and KIT. Both patients had PTEN genomic alterations and the PI3K/AKT/mTOR  pathway might play an important role in squamous cell transformation.Pathological transformation to squamous cell carcinoma after epidermal growth factor receptor ‐tyrosine kinase inhibitor (TKI) treatment has been reported, but details of the transformation remain unclear. Here, we report two cases with transformation to squamous cell carcinoma after treatment with EGFR‐TKIs. Both patients had PTEN genomic alterations and the PI3K/AKT/mTOR  pathway might play an important role in squamous cell transformation. EGFR) mutations show a high response rate of 60–70% to first‐ and second‐generation EGFR‐tyrosine kinase inhibitors (TKIs) EGFR and mesenchymal–epithelial transition proto‐oncogene amplifications and phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha gene (PIK3CA) mutations have also been reported as molecular resistance mechanisms Chemotherapy‐naïve patients with epidermal growth factor receptor (EGFR exon 19 insertion without T790M mutation. He received erlotinib (150 mg daily) and showed a partial response. After 28 months of therapy, the primary lung lesion started to advance. Transbronchial lung biopsy of the lesion showed adenosquamous carcinoma harbouring EGFR exon 19 insertion without EGFR T790M mutation , and atezolizumab (1200 mg on day 1 and every three weeks), then following maintenance therapy of atezolizumab (1200 mg every three weeks), and showed a partial response. After 14 months of therapy, the primary lung lesion worsened, and new lesions developed with bone metastases to the spine. Transbronchial lung biopsy of the lesion showed squamous cell carcinoma , PTEN c.963 del (p.N323Mfs*21), c.964_964 delA (p.N323Mfs*21), c.968 del (p.N323Mfs*21), PDGFRA c.2472C>T (p.V824V), and HRAS c.81T>C (p.H27H). PTEN expression was assessed immunohistochemically using the H‐score H‐score of PTEN expression was negative both before and after erlotinib therapy. Osimertinib (80 mg daily) was started and achieved stable disease over three months. Following disease progression with osimertinib, the patient was started on pembrolizumab (200 mg on day 1 and every three weeks) but after the first administration, the disease continued to progress. Then, S‐1 (100 mg/kg body weight on days 1–14 and every three weeks) was administered over three months. However, the patient died of lung cancer 58 months after diagnosis, and 17 months after transformation to squamous cell carcinoma.A 61‐year‐old man who was an ex‐smoker underwent computed tomography‐guided percutaneous lung biopsy. On the basis of the American Joint Committee on Cancer staging system, seventh edition, he was diagnosed as having stage IV (T2bN2M1a) adenocarcinoma of the lung  and showed a partial response. After nine months of this therapy, the primary lung lesion started to advance. Transbronchial lung biopsy of the lesion showed adenosquamous carcinoma , c.2369C>T (p.T790M), PTEN c.963 del (p.N323Mfs*21), c.964_964 delA (p.N323Mfs*21), c.968 del (p.N323Mfs*21), SMARCB1 c.1119‐41G>A (unknown), TP53 c.892G>T (p.E298*), and KIT c.1621A>C (p.M541L). H‐score of PTEN expression had converted from positive to negative after erlotinib therapy  adenocarcinoma of the lung  in osimertinib group, and zero of 140 (0%) in platinum plus pemetrexed group. The study showed that osimertinib resulted in significantly better progression‐free survival (PFS) than platinum‐pemetrexed  EGFR T790M mutation. Thus, the prognosis of lung cancer patients with squamous cell transformation and EGFR T790M mutation appears to be worse than that of patients with only EGFR T790M mutation. This is consistent with a report by Roca et al. Squamous cell transformation as a resistance mechanism to EGFR inhibitors has been investigated clinically. Roca et al. PTEN genomic alterations were identified in our patients with squamous cell transformation. In a study of surgically resected specimens, PTEN mutations were more often identified in ex‐smokers and in squamous cell carcinomas than in adenocarcinomas PTEN and PIK3CA in each two of four patients. Another case report by Kuiper et al. PIK3CA in a specimen after squamous cell transformation. Park et al. PI3K/AKT/mTOR pathway was activated by EGFR‐TKIs and loss of PTEN, which facilitates cell proliferation and resulted in squamous cell transformation. Interestingly, they found that one of four patients received everolimus  inhibitor) and showed radiological improvement. Further studies and more case reports are warranted to clarify the clinical utility of mTOR inhibitors in EGFR‐mutated patients with squamous cell transformation.At the molecular level, The present report has several limitations. First, the primary lung cancer before treatment with erlotinib therapy could have been adenosquamous cell carcinoma ab initio. We could not rule out this possibility because both patients were diagnosed using biopsy specimens. Second, we could not perform NGS analysis of the specimens before treatment with erlotinib, because the specimen volume was inadequate for analysis.EGFR mutation in lung adenocarcinoma with pathological transformation to squamous cell carcinoma. NGS analysis showed PTEN genomic alterations in both cases. Osimertinib was not fully effective in patients with squamous cell transformation, thus cytotoxic chemotherapies were probably better for these patients. Further studies and more case reports are warranted to elucidate the underlying mechanisms and investigate treatment modalities for patients with squamous cell transformation.In summary, we encountered two cases of Appropriate written informed consent was obtained for publication of this case report and accompanying images.


P < .05). Among these 13 hub‐genes, a four‐gene signature including neutrophil cytosol factor 2 , myosin‐If , sphingosine‐1‐phosphate receptor 4 , and ficolin‐1  alone or combined with two risk factors  may represent potential diagnostic biomarkers in obstructive CAD. Furthermore, the messenger RNA levels of NCF2, MYO1F, S1PR4, and FCN1 were higher in STEMI patients than that in stable CAD patients, although S1PR4 showed no statistical difference (P > .05). This four‐gene signature could also act as a prognostic biomarker to discriminate STEMI patients from stable CAD patients. These findings suggest a four‐gene signature  alone or combined with two risk factors  as a promising prognostic biomarker in the diagnosis of STEMI. Well‐designed cohort studies should be implemented to warrant the diagnostic value of these genes in clinical purpose.This study aims to explore the predictive noninvasive biomarker for obstructive coronary artery disease (CAD). By using the data set GSE90074, weighted gene co‐expression network analysis (WGCNA), and protein–protein interactive network, construction of differentially expressed genes in peripheral blood mononuclear cells was conducted to identify the most significant gene clusters associated with obstructive CAD. Univariate and multivariate stepwise logistic regression analyses and receiver operating characteristic analysis were used to predicate the diagnostic accuracy of biomarker candidates in the detection of obstructive CAD. Furthermore, functional prediction of candidate gene biomarkers was further confirmed in ST‐segment elevation myocardial infarction (STEMI) patients or stable CAD patients by using the datasets of GSE62646 and GSE59867. We found that the blue module discriminated by WGCNA contained 13 hub‐genes that could be independent risk factors for obstructive CAD ( Previous studies have suggested that the proinflammatory response plays critical roles in the pathogenesis of obstructive CAD, including both the innate and adaptive immune responses,Recently, Ravi et alTherefore, in this current study, differentially expressed genes (DEGs) in PBMCs between patients with obstructive CAD and free of obstructive CAD were identified, following by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Subsequently, through unsupervised hierarchical clustering and weighted gene co‐expression network analysis (WGCNA), the relationship between gene sets and phenotypes of CAD was ascertained. By using the bioinformatics methods and receiver operating characteristic (ROC) analysis, a potential four‐gene signature including ficolin‐1 (FCN1), myosin‐If (MYO1F), neutrophil cytosol factor 2 (NCF2), and sphingosine‐1‐phosphate receptor 4 (S1PR4), were further identified as biomarkers to discriminate obstructive CADs from nonobstructive CADs. Furthermore, the accuracy of this four‐gene signature was further explored for its accuracy to discriminate ST‐segment elevation myocardial infarction (STEMI) patients from stable CAD patients as well as may offer the potential novel therapeutic strategies for stable CADs.22.1In this study, the data set GSE90074 For the validation of the WGCNA findings in obstructive CAD, the gene expression data of PBMCs from STEMI patients were also retrieved from the NCBI GEO database. The data set, GSE62646, includes 14 PBMCs samples from stable CAD patients as control and 28 PBMCs samples from STEMI patients.2.2P‐values less than .05 was considered as a statistically significant DEG. The heatmap was visualized by using the heatmap package for “R” statistical software (version 3.3.3), as described previously.After normalizing the data, analysis of DEGs was performed using the package limma of R (version 3.3.3), and the gene with 2.3KEGG pathway and GO, including cellular component, molecular function, and biological process, were analyzed using the package clusterProfiler (version 3.2.14) of R (version 3.3.3), as described previously.2.42 = 0.8) were used to construct modules, and a threshold ≥0.7 was used to export network to Cytoscape .WGCNA package (version 1.60) in R was used to identify key modules based on the expression levels of DEGs in the data set GSE90074. The module is a cluster of closely interconnected genes, based on the dendrogram height. Modules were detected by using unsupervised clustering and dynamic branch cut methods .2.5https://string‐db.org/) was used to evaluate the protein–protein (PPI) network among genes in the enriched modules. PPI networks were constructed using Cytoscape (version 3.6.0), as described previously.Search Tool for the Retrieval of Interacting Genes/Proteins , as described previously.http://software.broadinstitute.org/gsea/msigdb/index.jsp). P < .05 was used as the cut‐off criterion.Gene set enrichment analysis (GSEA), including KEGG and GO enrichment analysis, is an effective method to compare the significant different priori defined sets of two groups. GSEA was performed with the gene expression of NCF2, MYO1F, S1PR4, and FCN1 in obstructive CAD patients using phenotype labels “high‐expression” vs “low‐expression” group by the GSEA software . Univariate and stepwise multivariate logistic regression (MLR) analyses were carried out to determine independent factors for the diagnosis of CAD by using the hub‐genes in the blue module and clinical risk factors. Receiver operating characteristic (ROC) curve analysis was used to calculate the value of area under the curve (AUC) for the selected genes and clinical risk factors to evaluate their predictive abilities for the diagnosis of CAD by using the package pROC, as described previously.33.1To find diagnostic biomarkers for obstructive CAD, we set up a workflow shown in Figure 3.2To search potentially key genes associated with obstructive CAD, we performed WGCNA with the most significant 2234 genes mentioned above to identify key modules of highly correlated genes. First, the hierarchical clustering tree (dendrogram) resulted in three significant modules with various colors, including blue, brown, and turquoise modules  analysis. We found that all the hub genes could be independent risk factors for obstructive CAD Table .P = .025), MYO1F (P = .001), S1PR4 (P = .015), and FCN1 (P = .012), were outstanding in the current stepwise MLR analysis, which had a significant association with prognosis of obstructive CAD. Next, we explored whether these four genes  were associated with obstructive CAD by performing ROC curve analysis in the same population. We found that the AUC was 0.606 for FCN1, 0.604 for MYO1F, 0.648 for NCF2, 0.626 for S1PR4, and 0.700 for the combination of these four genes . Taken together, these results demonstrate a high diagnostic accuracy of four‐gene signature as a novel biomarker for obstructive CAD.Then, stepwise MLR analysis showed that four genes showed the lowest Akaike information criterion (AIC) value, which could remove other confounding factors and got the best result of data fitting. Four genes, including NCF2 .As we know, CAD has many clinical risk factors, including age, sex, BMI, hypertension, hyperlipidemia, diabetes mellitus, and the degree of coronary artery obstruction. Then, further study was explored to analyze the relationship between the four genes and these CAD risk factors. Then, the results of ULR analyses showed that sex and hyperlipidemia were significantly associated with the diagnosis of CAD, but not with the other risk factors Table . The AUCP = .025, .001, .015, .012, and .010, respectively; Figure As indicated above, the result of stepwise MLR analysis showed that the combination of clinical risk factors with the four genes with a lower AIC value indicated an association between NCF2, MYO1F, S1PR4, and FCN1 expression levels in PBMCs and male sex, and hyperlipidemia Table . To deteGiven the similar diagnostic values for the genes NCF2, MYO1F, S1PR4, and FCN1 in obstructive CAD, Spearman‐rank correlation was used to analyze the genes that correlated with the obstructive CAD and CAD severity. The results indicated that these four genes were correlated with one another significantly, but not correlated with the other risk factors Figure F. All th3.5P < .001; Figure STEMI is the significant risk factor for obstructive CAD.3.6In the functional enrichment analysis of four‐finding genes, we divided the 93 obstructive CAD patients into two groups due to the expression levels of NCF2, MYO1F, S1PR4, or FCN1 (high‐expression group vs low‐expression group) and applied GSEA analysis to compare the different pathways between the two groups. Our finding showed that viral myocarditis,4Decades of research have provided a deep understanding of the etiology of obstructive CAD, however, the predictive biomarkers, especially noninvasive biomarkers, for obstructive CAD diagnosis, are still limited.In this study, by deeply and systemically reanalyzing the GSE90074 data set, KEGG and GO analyses of total DEG demonstrate that during the development of obstructive CAD, immune system cells activated in the plaque, which is consistent with previous findings.Further validation of the diagnostic accuracy of NCF2, MYO1F, S1PR4, and FCN1 in STEMI patients showed that these four hub genes could also act as accurate biomarkers to discriminate STEMI patients from stable CAD patients. The expression levels of NCF2, MYO1F, S1PR4, and FCN1 were relatively higher in STEMI patients than in stable CAD patients. NCF2 is a component of the leukocyte NADPH oxidase complex that produces superoxide. Accumulating evidence has indicated that NCF2 plays critical roles in the development of autoimmune diseases, such as inflammatory bowel diseases, systemic lupus erythematosus,In conclusion, a four‐gene signature  could act as a noninvasive diagnostic biomarker for obstructive CAD. In combination with the risk factor, sex, and hyperlipidemia, it could improve the diagnostic accuracy of distinguishing obstructive CAD from free of obstructive CAD. Therefore, our study contributed a new potential noninvasive biomarker for obstructive CAD. Undoubtedly, future well‐accepted clinical studies with larger samples size, standardized protocols, and more homogenized populations would be needed to fully research the prognostics potential of this four‐gene signature in patients with obstructive CAD.The authors declare that there are no conflict of interests.Xian‐Gang Mo designed experiments, analyzed data, and wrote the manuscript; Wei Liu, Yao Yang, Saber Imani, Shan Lu, Guorong Dan, Xuqiang Nie, Jun Yan, and Rixing Zhan designed experiments and analyzed data; Xiaohui Li revised the manuscript; Bingbo Chen and Yue Cai designed experiments, analyzed data, and edited the manuscript; Youcai Deng devised the concept, designed the research, supervised the study, and wrote the paper.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.Supporting informationClick here for additional data file.