The Western diet (WD) and hyperlipidemia are risk factors for vascular disease, dementia, and cognitive impairment. However, the molecular mechanisms are poorly understood. This pilot study investigated the genomic pathways by which the WD and hyperlipidemia regulate gene expression in brain microvessels. Five-week-old C57BL/6J wild type (WT) control and low-density lipoprotein receptor deficient (LDL-R−/−) male mice were fed the WD for eight weeks. Differential gene expression, gene networks and pathways, transcription factors, and non-protein coding RNAs were evaluated by a genome-wide microarray and bioinformatics analysis of laser-captured hippocampal microvessels. The WD resulted in the differential expression of 1972 genes. Much of the differentially expressed gene (DEG) was attributable to the differential regulation of cell signaling proteins and their transcription factors, approximately 4% was attributable to the differential expression of miRNAs, and 10% was due to other non-protein coding RNAs, primarily long non-coding RNAs (lncRNAs) and small nucleolar RNAs (snoRNAs) not previously described to be modified by the WD. Lipotoxic injury resulted in complex and multilevel molecular regulation of the hippocampal microvasculature involving transcriptional and post-transcriptional regulation and may provide a molecular basis for a better understanding of hyperlipidemia-associated dementia risk. Alzheimer’s disease (AD) is a progressive disease characterized by a decline in cognitive function and loss of memory, and its etiology includes both environmental and genetic factors . The strThe global increase in obesity has been linked to a decline in complex carbohydrate and fiber intake, diets with fewer fruits and vegetables, and a shift towards diets high in fat and refined sugars—otherwise known as the Western-type diet (WD). An association has been reported between obesity and cognitive dysfunction and reduced neural integrity  . One conHigh dietary fat and cholesterol also have functional sequelae in the brain. The blood–brain barrier (BBB) plays a key role in many cognitive dysfunction and neurodegenerative disorders, presumably leading to increased movement of immune cells and immune intermediaries into the brain, which contribute to neuro-inflammation and consequently, neuro-degeneration ,9. The BIn vivo studies have been performed on whole brains or specific regions of the brain, thus obscuring the specific effects of a hyperlipidemic diet on endothelial cells. In addition, it is known that lipids are central to the pathogenesis of dementia, yet the multifactorial mechanisms by which they contribute to cognitive dysfunction in the brain are not entirely understood. Several studies have employed genomic approaches to assess for molecular level effects following the Western-type diet or hyperlipidemia induced by the low-density lipoprotein receptor-deficient (LDLR−/−) genotype. For example, high-fat diets can modulate the expression of genes related to neuronal projections and synaptic transmission corresponding to significant deterioration of neurite morphology and cognition . In addiMore recently, it has also been shown that a high-fat diet can also regulate long non-coding RNAs (ncRNAs) . LncRNAsLDL-R-deficient mice are widely used as models for the investigation of atherosclerosis and diet-associated lipotoxic injury because the LDL receptor is essential to the removal of ApoE-containing lipoproteins from the blood . DeficieThus, the goal of our study was to determine and characterize the molecular mechanisms for the genomic effects of a high-fat diet and experimental hyperlipidemia on brain endothelium of LDL-R-deficient male mice, by performing global transcriptomic analyses on laser-captured isolated microvessels from the hippocampal regions of the brain. In addition, most prior studies have evaluated the impact of lipids on one type of RNA but have not ventured beyond that or looked at potential interactions, and therefore, a secondary goal was to perform integrated omics analyses to better understand the complexity of genetic regulation by non-protein-coding RNAs in response to the WD in males.w/w) for 8 weeks. There were four experimental treatment groups randomly assigned to the diets: WT fed CD = WT-CD, WT fed WD = WT-WD, LDL-R −/− fed CD = LR-CD, and LDL-R fed WD = LR-WD; n = 7 mice/group. Animals were housed in single cages in a temperature- and humidity-controlled environment with a 12 h light/dark cycle in the University of California, Davis Mouse Biology Program. Body weight was measured at baseline and at the completion of the dietary intervention period, and activity and food intake monitored daily by vivarium staff. Research was conducted in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animals. The institutional review board of the University of California, Davis, the Institutional Animal Care and Use Committee (IACUC) approved this project protocol number 19750 on 7 February 2017.Low-density lipoprotein receptor-deficient  and C57BL/6J wild type  male mice were fed either a standard chow control diet  or a Western diet  composed of 21% fat and 0.2% cholesterol (Fasting lipid levels were measured in serum samples that were stored at −80 °C until assayed. Triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) were measured using enzymatic assays from Fisher Diagnostics , and precipitation separation from AbCam  adapted to a microplate format. Fasting glucose and insulin levels were also measured on serum samples. Glucose was measured using enzymatic assays from Fisher Diagnostics , and insulin was determined by electrochemiluminescence from Meso Scale Discovery  according to the manufacturer’s instructions. All assays were performed by the UC Davis Mouse Metabolic Phenotyping Center (MMPC) in triplicate, on non-pooled plasma samples.later®-ICE  to prevent RNA degradation, and stored at −80 °C until use. When ready for use, cryosections from the hippocampal segments were submerged in nuclease-free water and dehydrated in desiccant.Following completion of the dietary feeding period, mice were anesthetized by intraperitoneal xylazine/ketamine and euthanized by exsanguination during the light phase of their light/dark cycle, then intravascularly perfused with DEPC-treated PBS. Intact brains were rapidly removed under RNAse free conditions, cut into regions including the temporal lobe segment, and embedded using HistoPrep Frozen Tissue Embedding Media . To identify the hippocampus and hippocampal neurons, brain sections in the medial aspect of the temporal lobe were stained with hematoxylin and visualized with microscopy by a histopathology expert at UC Davis (Dr. Dennis Wilson). The hippocampus was then coronally cryosectioned . Hippocampal cryosections were placed on charged RNA-free PEN membrane glass slides, treated with RNAFor the analysis of the gene transcriptome of the hippocampal brain microvessels, endothelial microvessels (<20µm) were first identified in the hippocampal brain cryosections by alkaline phosphatase staining utilizing 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chloride (BCIP/NBT) substrate as previously described . Laser cTotal RNA was extracted from the laser-captured hippocampal brain microvessels (100 microvessels/sample) from each of the four experimental animal groups using an Arcturus PicoPure™ RNA Isolation Kit  according to the manufacturer’s instructions. The quality of the RNA from the LCM-derived vessels was assessed by Nanodrop, and RNA integrity verified by qRT-PCR of the control gene transcription (GAPDH). RNA quantification was performed according to Affymetrix RNA quantification kit with the SYBR Green I and ROX™ Passive Reference Dye protocol .® WT Pico Kit. SscDNA (5.5 µg) was fragmented by uracil-DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) and labeled by terminal deoxynucleotidyl transferase (TdT) using the DNA labeling reagent that is covalently linked to biotin. Fragmented and labelled ssCDNA samples in triplicate were then submitted to the UC Davis Genome Center shared resource core for hybridization, staining, and scanning using the Affymetrix WT array hybridization protocol following the manufacturer’s protocol. Hybridization of the fragmented and labelled ssCDNA samples was done using GeneChip ™Hybridization Oven 645, and samples then washed and stained using GeneChip ™ Fluidics Station 450. The arrays were scanned using a GeneChip™ Scanner 3000 7G . Quality control of the microarrays was done using the Affymetrix Expression Console software version 1.4.1 and data analysis performed using the Affymetrix Transcriptome Analysis Console software version 3.1.0.5.For the transcriptomics analysis, we used Affymetrix GeneChip Mouse Gene 2.0 ST Array . RNA (125 pg) was used to prepare cRNA and sscDNA using an Affymetrix GeneChip ® Green Supermix as the fluorescent reporter . Specific primers were designed with the Primer3 software [To corroborate the microarray analysis results, we performed qRT-PCR on 11 randomly selected differentially expressed RNA transcripts. For these experiments, RNA (75 ng) from the laser-captured brain microvessels was reverse-transcribed into cDNA using iScript Reverse Transcription Supermix for RT-Qpcr . qRT-PCR for selected genes was performed in the ABI Vii7 Sequence detection system . Reactions were carried out in 384-well optical plates containing 25 ng cDNA/well and SsoAdvanced™ Universal SYBR software  using thBioinformatics analysis of differentially expressed genes was performed by two of the study investigators (SN and DM) using multiple software tools. We compared each study group  to the control (WT-CD). For the fold change calculations, it was also necessary to input the experimental group data and compare them to the control group data. This information is required by the microarray software  used in the project.https://david.ncifcrf.gov/home.jsp) [http://revigo.irb.hr/) [https://genetrail2bioinfuni-sbde) [https://portalgenegocom) to identify significantly over represented pathways. Enrichment statistics were calculated for these data sets assuming a hypergeometric distribution to identify significantly over represented pathways. Gene network and transcription factor analyses were performed using Metacore™. MicroRNA validated targets were searched using the miRWalk database  [http://www.atgc-montpellier.fr/permutmatrix/) [http://bioinfogp.cnb.csic.es/tools/venny/). Network analysis of interactions between functional groups was identified using Metascape (http://metascape.org/) [https://cytoscape.org/) [http://wwwmetaboanalystca/MetaboAnalyst/faces/homexhtml) [http://starbase.sysu.edu.cn/index.php) [http://www.mircode.org/index.php) [https://rnacentral.org) [Gene ontology of identified differentially expressed genes was done using the David bioinformatics database (ome.jsp) ,22, and irb.hr/) . Canonicni-sbde)  and the ex.html)  that enamatrix/) . Venn dipe.org/) , and obtpe.org/) . OmicsNemexhtml)  was useddex.php) , starBasdex.php) , and theral.org) .p < 0.05 and a ±2.0-fold change were considered as significantly differentially expressed. Mean body weight and plasma lipid levels of all diet/genotype groups  were expressed as means ± standard error of the mean (SEM), and significance was determined at p ≤ 0.05 using unpaired student’s t-tests . qRT-PCR determined the gene expression in the hippocampal microvessels of the experimental mice, compared with WT-CD, and was expressed as a log2-fold change, and statistical significance was determined by unpaired student’s t-tests .For the microarray, a two-way ANOVA  was used for the statistical analysis of the microvessel transcriptome of the WT-WD, LR-CD mice, and LR-WD, each compared with the WT-CD mice. All genes from the microarray with p < 0.05 respectively for all group comparisons): The dietary treatment resulted in the expected weight gain in the study mice as follows: mean weight for WT mice at baseline was 21 g and increased by an average of 24% when fed with CD and 39% when fed with WD; mean weight of LR mice at baseline was 17.25 g and increased by an average of 62% when fed with CD and 74% when fed with WD (p < 0.05), respectively, and 285.6 and 1151.8 mg/dL (p < 0.05) for the LR-CD and LR-WD-fed mice, respectively: p < 0.05) in the LR-WD-fed groups: p < 0.05) in the LDL-R−/− genotype and WD-fed mice compared with the WT mice on the CD. These results are consistent with what has been published previously for these experimental models [After eight weeks on the experimental diets, the mean total cholesterol levels in the CD- and WD-fed WT mice were 89.3 and 252.8 mg/dL  were differentially expressed (DE). Volcano plots of the significantly up- or down-regulated genes in the brain microvessels of WT-WD showed up-regulation of two genes and down-regulation of eight genes compared with the microvessels of the WT-CD mice: A random sample of eleven differentially expressed protein-coding and ncRNA, representative of each of the experimental genotype/diet groups, was tested by qRT-PCR and confirmed to have the same direction of change in gene expression (up- or down-regulation) as observed with the microarrays . These dTo identify the biological functions of differentially expressed protein-coding genes, and consequently the potential cellular processes which could be affected by changes in their expression, we performed a series of bioinformatic analyses. Our first analysis was to identify the gene ontologies of the differentially expressed genes. The genes identified as differentially expressed are involved in many biological functions: Next, we performed gene network analyses using a text-mining algorithm (MetaCore) to identify the functional groups of the gene networks . Using tFollowing the gene network analyses, we used KEGG and MetaCore to identify the cellular pathways for the differentially expressed genes. We observed the differential regulation of a number of important cellular pathways including those for neurological function-related pathways , cellular metabolism , cell signaling , endothelial function , and a few other cellular processes . In geneUsing the ClueGo tool on Cytoscape, we also searched cellular pathways that form networks to identify the function of the groups of pathways of differentially expressed genes. Using this approach, we identified over 50 pathways in a network. These networks of pathways formed functional groups involved in cell signaling, oxidative stress, endothelial cell function, and neurofunction.We also performed bioinformatics analyses of the gene expression data to identify potential transcription factors (TF) whose activity could be modulated by lipid injury and be involved in mediating the observed genomic effects. The most statistically significant transcription factors were ETS1 (ETS proto-oncogene 1), c-Myc (cellular myelocytomatosis), FOXP3 (forkhead box P3), and GABPalpha (GA binding protein transcription factor) . CompariOur microarray analysis allowed us to study not only protein-coding but also miRNAs. Using this approach, we identified that lipotoxic injury can modulate the expression of miRNAs in brain endothelial cells . We idenTo identify potential target genes for the differentially expressed miRNAs in the study groups, we used the miRWalk database . When coOur next step was to identify the pathways of the target genes of the differentially expressed miRNAs. We used the miRWalk online tool to search for the pathways in the KEGG database. Using this approach, we showed that compared with the WT-CD mice, there were 6, 68, and 25 pathways of miRNA target genes in the WT-WD, LR-CD, and WD-fed LDL-R−/− mice, respectively . Among tThe comparisons of the pathways identified with the differentially expressed genes and pathways identified using the target genes of miRNAs revealed a group of pathways in common between the two modes of gene regulation . Among tWe used the OmicsNet tool to incorporate the data obtained by the individual analyses and integrate it with the data obtained on the effect of lipotoxic injury on the expression of protein-coding genes, potential transcription factors involved in the observed genomic response, and potential post-transcriptional regulation of gene expression . We obseAs detailed above, most of the differential gene expression in our study system was attributable to the differential regulation of cell signaling proteins and their transcription factors. However, approximately 4% of the differential expression was attributable to the differential expression of miRNAs, and 10% was due to other ncRNA, primarily long non-coding RNAs (lncRNAs) and small nucleolar RNAs (snoRNAs).Regarding non-coding RNAs, we identified the differential expression of a total of 109 lncRNAs and 97 snoRNAs in the experimental groups when compared with the control WT-CD mice: The next step in our bioinformatic analyses was to integrate the different omic analyses together. Using a heat map tool, we first compared the pathways identified from the three omic analyses, i.e., from differentially expressed protein-coding genes, targets of differentially expressed miRNAs, and targets of lncRNAs . Using tThis pilot study was a large-scale transcriptome gene-profiling of the male mice hippocampal microvasculature in relation to lipotoxic injury from two different sources, diet and genetics, using the Western diet (WD) and/or LDL-R−/− (LR) genotype, respectively. Our study focused on the molecular mechanisms of differential gene expression in the brain microvessels due to their significance in the pathogenesis of vascular dementia, and also due to our recently published work demonstrating that the WD results in increased blood–brain barrier (BBB) permeability and cognitive impairment , providi➢The WD resulted in the differential expression (primarily up-regulation) of a large number of genes (1972), representing 5.7% of the genome of microvessels in the hippocampus of male mice;➢Overall, the differential gene expression was associated with the differential regulation of cell signaling proteins and their transcription factors, with complex mechanisms of action for genes that regulate increased endothelial dysfunction following lipid stress as the main disruption, particularly via pathways that serve to increase permeability, consistent with the previously reported increase in BBB permeability following the WD;➢There were some differences in the differential gene expression for diet and genotype. Differentially expressed genes involved in focal adhesion, ECM–receptor interaction, and signaling pathways  were up-regulated with lipid injury in the LR genotype while down-regulated by the WD in the wild type mice;➢Most of the differential gene expression was attributable to protein-coding genes (85%), but approximately 4% was due to the differential expression of miRNAs, and 10% was due to other non-protein-coding RNAs not previously found to be affected by the WD, including mostly long non-coding RNAs (lncRNAs) and small nucleolar RNAs (snoRNAs). The targets of lncRNAs included genes involved in NF-kB signaling, Ras/Rap signaling, focal adhesion, actin cytoskeleton organization, cell adhesion, chemokine signaling, tight junctions, and adherent junctions;➢Lipotoxic injury resulted in previously unreported complex and multilevel molecular regulation of the hippocampal microvasculature involving transcriptional and post-transcriptional regulation. Post-transcriptional regulation accounted for up to a third of the differential gene expression;➢Specific detailed examples of this complex regulation for the representative genes, pathways, transcription factors, and non-coding RNAs are provided below.Our main findings are as follows:Our study demonstrated the expected significant differences in cholesterol and lipid levels between the control diet (CD) and the high-fat LR-WD in male mice. The LR-CD mice spontaneously demonstrated hyperlipidemia because of the absence of the LDL receptor. While the physiological implications of severe hyperlipidemia are a topic of further study, it has been shown to correlate with accelerated atherosclerosis and vascular injury in other systems . We deci). This observation suggests that lipotoxicity induced by the LR genotype has a potentially greater impact on differential gene expression than the WD and could mask the effect of the WD in the LR genotype.However, it was previously unknown how a high-fat diet affects the transcriptome of the brain hippocampal microvessels in males. Using a candidate gene approach in our previous work, we identified activating transcription factor 3 (ATF3) as an important regulator of neuro-inflammation in male mice . The preSupplemental Figure S7It has previously been suggested that consumption of fatty acids can modulate gene expression in rat brain  and thatAmongst the differentially expressed genes controlling BBB specifically was GTPase HRas, a GTP-binding protein that plays an important role in cytoskeletal reorganization, cell polarity, cell cycle progression, or angiogenesis . InadequWe also observed that lipotoxic injury differentially regulates the expression of genes involved in pathways related to neurofunction and serotonergic pathways. Among these genes is MOAB, which encodes for Monoamine oxidases isoform B, important in oxidative deamination and thus vascular oxidative stress and endothelial dysfunction . In our Using bioinformatic tools, we identified from the list of differentially expressed genes potential transcription factors (TFs) that could have their activity modulated by lipotoxic injury and be involved in the observed genomic effect. Among the TFs identified, 17 were in common between the study groups. Among them was hepatocyte nuclear factor 4 alpha (HNF4a), whose activity is known to be modulated by fatty acids . HNF4a cIn addition to assessing the effect of chronic consumption of the WD on the expression of protein-coding genes, we also identified a previously unknown effect of lipid injury on the expression of ncRNA in the male hippocampal microvasculature. These included microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), and long non-coding RNAs (lncRNAs). This is of significance as ncRNAs have been previously reported to play a vital role in cognition and the vasculature, but the mechanisms have not been defined.Following a high-fat diet, miRNAs have been reported to be involved in neural function and in metabolic and inflammatory pathways that play a role in atherogenesis . In our Additionally, some several hundred target genes were identified for each dietary experimental group from the analyses of the predicted miRNA target genes. Interestingly, we observed that 28% of the miRNA target genes overlapped with differentially expressed genes (DEG), suggesting that nearly one-third of the gene expression could be regulated at the post-transcriptional level by lipid injury, while the remaining can be regulated at the level of transcription. The target genes of the differentially expressed miRNAs were involved in pathways related to endothelial cell permeability and function, cell signaling, insulin signaling/resistance, apoptosis, and dementia. Insulin dysregulation has been linked with cognitive dysfunction  and endoOur microarray analysis also identified the differential expression of other ncRNA other than miRNAs, including snoRNAs and lncRNAs. SnoRNAs play a key role in the regulation of gene expression, post-transcriptional modification of other RNAs, and in stabilizing the genome . We obseThis study also revealed that lipotoxic injury can control the expression of several lncRNAs. LncRNAs are regulatory RNAs involved in the transcriptional, post-transcriptional, and translational modulation of genes. As such, lncRNAs function in several aging-related processes, including apoptosis, neuronal differentiation, and immune or stress responses ,59. We oIn summary, to our knowledge, these findings are novel indicators that lipotoxic injury modulates the expression of non-coding RNAs  in the male hippocampal microvasculature via targets that may play an important role in lipotoxic injury-associated brain microvascular disease and vascular dementia.Integrative analyses of the genomic data allowed us to identify the full complexity of the genomic effects of lipotoxicity in the brain microvasculature. Bioinformatic analyses of the genes allowed us to identify the cellular functional pathways in which they were involved, including angiogenesis, apoptosis, and immune cell interactions as well as functional networks, including for cell adhesion and signal transduction. The comparison of the pathways identified separately for the different types of RNAs showed that a third of them were in common for at least two different RNA types and 12 of them were identified for the three different RNA types studied . This obOur study is one of a few studies that have analyzed multi-omic regulation simultaneously and integrated different types of RNAs in the brain microvasculature ,70, and In alignment with our hypothesis, our results showed profound transcriptome changes in response to the WD in the hippocampal microvasculature of male mice including the modulation of protein-coding genes, miRNAs, snoRNAs, and lncRNAs, as well as the corresponding cellular functional pathways, and the mechanism of regulation by transcription factors. Integrative analyses of the genomic data also identified the cellular functions primarily affected by lipid-associated vascular injury to include angiogenesis, apoptosis, immune cell interactions, and gene regulation and RNA biogenesis, which could be related to vascular injury or response to lipid injury. Among these gene networks and pathways, endothelial cell adhesion, cytoskeleton organization, neurofunction, cell junctions and chemotaxis, and focal adhesion are regulated—mechanisms involved in the regulation of endothelial permeability. This is of significance because a disruption to the endothelial permeability in the brain, where endothelial cells are part of a neurovascular unit, results in BBB dysfunction, which may be a significant contributor to the pathogenesis of cognitive impairment, Alzheimer’s disease, and other related dementias. As such, our work helps to significantly advance the field and elucidate the molecular mechanisms whereby the Western diet disrupts brain microvascular endothelial function and how that may predispose to vascular dementia. Future work will need to address whether and how the high-glycemic component of a Western diet contributes to microvascular lipid injury in the brain, the reversibility of diet associated vascular dementia, and the interaction of genetic factors and diet on vascular dementia.


Ophiognomonia clavigignenti-juglandacearum (Oc-j) is a plant pathogenic fungus that causes canker and branch dieback diseases in the hardwood tree butternut, Juglans cinerea. Oc-j is a member of the order of Diaporthales, which includes many other plant pathogenic species, several of which also infect hardwood tree species. In this study, we sequenced the genome of Oc-j and achieved a high-quality assembly and delineated its phylogeny within the Diaporthales order using a genome-wide multi-gene approach. We also further examined multiple gene families that might be involved in plant pathogenicity and degradation of complex biomass, which are relevant to a pathogenic life-style in a tree host. We found that the Oc-j genome contains a greater number of genes in these gene families compared to other species in the Diaporthales. These gene families include secreted CAZymes, kinases, cytochrome P450, efflux pumps, and secondary metabolism gene clusters. The large numbers of these genes provide Oc-j with an arsenal to cope with the specific ecological niche as a pathogen of the butternut tree. Ophiognomonia clavigignenti-juglandacearum (Oc-j) is an Ascomycetous fungus in the family Gnomoniaceae and order Diaporthales. Like many of the other species within the Diaporthales, Oc-j is a canker pathogen, and is known to infect the hardwood butternut (Juglans cinerea) (Discula destructiva), butternut canker (Ophiognomonia clavigignenti-juglandacearum), apple canker , Eucalyptus canker , and perhaps the most infamous and well-known chestnut blight (Cryphonectira parastica). Furthermore, several species in the Diaporthales also cause important disease of crops including soybean canker , soybean seed decay (Diaporthe langicolla) and sunflower stem canker (Diaporthe helianthi). In addition to pathogens, there are also a multitude of species that have a saprotrophic or endophytic life strategy (cinerea) . The Diacinerea) , which icinerea) . Numeroustrategy . HoweverOc-j is an invasive species, introduced into the U.S. from an unknown origin. This introduction caused extensive damage among the butternut population in North America during the latter half of the 20th century. The first report of butternut canker was in Wisconsin in 1967 (Sirococcus clavigignenti-juglandacearum (Sc-j) (Ophiognomonia and was reclassified as Ophiognomonia clavigignenti-juglandacearum (Oc-j)  . Phylogem (Oc-j) . The sud America .Oc-j is a devastating pathogen of the butternut, Juglans cinerea, there are also several species in the genus Ophiognomonia that are endophytes or saprophytes of tree species in the order Fagales and more specifically the Juglandaceae or walnut family (Sirococcus (Ophiognomonia) clavigignenti-juglandacearum as an endophyte of Acer truncatum, which is a maple species native to northern China  isolated from an infected butternut tree in Wisconsin in 1978, was sequenced. For DNA extraction, isolates were grown on cellophane-covered potato dextrose agar for 7–10 d, and mycelia was collected and lyophilized. DNA was extracted from lyophilized mycelia using the CTAB method as outlined by the Joint Genome Institute for whole genome sequencing . The total DNA quantity and quality were measured using both Nanodrop and Qubit, and the sample was sent to the Hubbard Center for Genome Studies at the University of New Hampshire, Durham, New Hampshire. DNA libraries were prepared using the paired-end Illumina Truseq sample preparation kit, and were sequenced on an Illumina HiSeq 2500.For this study, the ex-type culture of We corrected our raw reads using BLESS 0.16  with thePRJNA434132. Gene annotation was performed using the MAKER2 pipeline  and Diaporthe helianthi . Proteins that both contain a signal peptide and are predicted to be a CAZyme are annotated as a secreted CAZyme.SignalP  was usedOc-j contains a total of 52.6 Mb and 5,401 contigs, with an N50 of 151 Kbp  were missing. This result indicates that the Oc-j genome is relatively complete.The draft genome assembly of  151 Kbp . The com 151 Kbp . Out of Cryphonectria parasitica (jgi.doe.gov), Chrysoporthe cubensis  were used. These species include: cubensis , Chrysopcubensis , Chrysopafricana , Valsa mlsa mali , Valsa plsa pyri , Diaportampelina , Diaportspalathi , Diaportngicolla , Diaportelianthi , and Mela crassa  and Magne grisea . We show species , in whicOc-j gene repertoire, the PFAM domains were identified in the protein sequences . Oc-j contains 29 genes with this domain, while N. crassa has none and the other related species have between 3 and 8 genes  are a superfamily of monooxygenases that play a wide range of roles in metabolism and adaptation to ecological niches in fungi . Due to 223 CYPs , more thectively . Accordiectively , very feectively . The oneN. crassa to 51 genes in V. mali, while Oc-j has 47 genes , followed by non-ribosomal peptide synthetases  and terpene synthases (9)  .Oc-j has the third highest number of clusters, only after D. longicolla and Melanconium sp.; however, when normalized by total gene number in each species, Oc-j is shown to have 6.4 secondary metabolism gene clusters per 1,000 genes, the highest among all species included in this study. The D. helianthi genome contains only six clusters, likely due to the poor quality of the genome assembly with an N50 of ~6 kb . Future research will need to focus on understanding the prevalence of these genes associated with complex biomass degradation among other members of the Ophiognomonia genus which include endophytes, saprophytes and pathogens. It will be interesting to know which of these different classes of genes are important in the evolution of distinct fungal lifestyles and niche adaptation. Future research will also focus on comparing the butternut canker pathogen to canker pathogens of other tree species which produce large numbers of secondary metabolites known to be important in host defense. Our genome also serves as an essential resource for the Oc-j research community.We constructed a high-quality genome assembly for 10.7717/peerj.9265/supp-1Supplemental Information 1Click here for additional data file.


Using structural equation modeling, wetested hypothesized direct and indirect effects of race/ethnicity, SES, andthree psychosocial mediational variables  on two outcome variables—psychologicaland social well-being—assessed at 1-year follow-up. Our model indicated that:(1) race/ethnicity and SES were significantly associated with each other andwith each psychosocial mediator; (2) higher SES was directly and indirectlyassociated with both measures of well-being; and (3) collective self-esteem andeveryday discrimination mediated the association between SES and both measuresof well-being. The model also indicated that racial/ethnic associations withpsychological mediators and outcomes are evident in the context of SES, butthese effects might be suppressed when the model does not consider SES. Findingshighlight the critical role of SES and race/ethnicity in determining thepsychological and social well-being of sexual minority men. Specification ofmediating variables—collective self-efficacy, everyday discrimination,internalized homophobia—indicates potential intervention targets to improvepsychological and social health in sexual minority men. Associations betweenrace/ethnicity and SES support the need for intersectional frameworks inaddressing the health of sexual minority men.A large body of research demonstrates disparities in psychological healthattributed to sexual minority identity, racial/ethnic minority identity, andsocioeconomic status (SES). Fewer studies have explicated the role of thesemultiple attributes on psychological health and explored the role of SES andpsychosocial resources in determining outcomes. We analyzed data from ProjectSTRIDE, a longitudinal survey involving a diverse sample of gay and bisexualadult men ( Socioeconomic status (SES) is a well-established predictor of well-being, includingphysical and mental health . StudiesIn addition to SES, race/ethnicity remains a prominent factor explaining physical andmental health disparities in heterosexual and non-heterosexual populations . GayandOur study seeks to reframe health disparities by examining pathways that may explain therelationship between sexual and racial/ethnic minorities and psychological health. Inthis study, we started to investigate potential pathways that heighten risk for adversehealth outcomes among individuals with multiple minority identities, including Black andLatinx GBM. This approach recognizes the intersection of sexual orientation andrace/ethnicity, which has been the focus of an emerging body of literature onintersectionality . Guided This study begins to address evidence gaps by simultaneously examining the association ofSES, race/ethnicity, and well-being in a longitudinal cohort study of gay and bisexualmales. We aim to investigate the direct and indirect associations of race/ethnicity withpsychosocial resources and direct and indirect associations of SES with psychologicaland social well-being. We are interested in empirically testing the followingrelationships: (1) direct and indirect association of SES with well-being and (2) directand indirect association of race/ethnicity with psychosocial resources. In this study,we assessed the indirect association of race/ethnicity via SES on psychosocial resourcesand the indirect effect of SES via psychosocial resources on well-being. Outcomes wereassessed at 1-year follow-up. Consistent with the literature , we examData came from Project STRIDE: Stress, Identity, and Mental Health, a largeNational Institute of Mental Health (NIMH)-funded longitudinal research studyconducted in the New York City area . A detain = 524) were recruited usingvenue-based and snowball sampling methods to ensure diversity of respondentsbased on gender, sexual orientation, race/ethnicity, and age. All respondentswere recruited in person by research workers who approached potential studyparticipants in the sampling venues. A cap of 25% was established for the numberof respondents taken from the five venue types: bars, non-bar establishments,outdoors, groups, and events. Snowball sampling was used to recruit respondentswho were less likely to be identified in these venues and to increase thediversity of the study sample. Respondents recruited in public venues were givenletters of invitation to pass along to their friends and colleagues. At eachvenue, research staff explained the study and its activities to potentialparticipants, who then filled out a screening form to determine eligibilityself-identified as cis-gender male or female and were assigned that sex atbirth; (2) self-identified as lesbian, gay, bisexual (LGB), straight, or usedother terms conveying such identification ; (3)self-identified as White, Black, or Latino or used other terms conveying suchidentifications ; (4) were between the ages of18 and 59; (5) resided in New York City for two years or more; and (6) were ableto speak English well enough to engage in casual conversation .IndividSES referred to respondents’ access to social and economic resources atbaseline. We operationalized SES as a latent variable, given that there isno single best-observed indicator of SES . To incrWell-being referred to two types: psychological and social. Both variableswere assessed at 1-year follow-up. Psychological or personal well-beingassessed the respondents’ perception of various aspects of theirpsychological well-being, including autonomy, environmental mastery,personal growth, positive relations with others, purpose in life, andself-acceptance . This ouWe included three types of psychosocial mediators that have been identifiedto correlate directly with race/ethnicity, SES, and well-being . Thesemoften) to 4 (never). Itemsincluded: “How often have you been called names or insulted?” and “Howoften have you experienced people acting as if they are afraid of you?”Responses were recoded so that higher scores reflected the frequentexperience of discrimination.Everyday discrimination referred to respondents’ experience of chronicand routine unfair treatment. often) to 4 (never). Sample itemsincluded: “How often have you wished you were not gay?” and “How oftenhave you wished that you could develop more erotic feelings toward theopposite sex?” Responses were recoded so that higher scores indicatedgreater internalized homophobia.Internalized homophobia evaluated the extent to which gay and bisexualmales do not accept their sexual orientation, are uneasy about theirsame-sex desires, and seek to avoid homosexual feelings . This vastrongly agree) to 7(strongly disagree). Items included, “I am a worthymember of the social groups I belong to” and “Overall, my social groupsare considered good by others.” Responses were recoded so that higherscores indicated a higher level of collective self-esteem.Collective self-esteem assessed respondents’ evaluation of theircollective identity and group memberships .This vaWe included three baseline demographic variables in our structural equationmodel: race/ethnicity , country of birth (theUnited States or not in the United States), and household size, defined asthe number of people in the household. Country of birth and household sizewere added as covariates when we examined the relationship betweenrace/ethnicity and SES.2 test, whichis an appropriate measure of fit for models with up to 200 cases . 00 cases . Wealso00 cases .2 test, RMSEA, CFI, and TLI). As for indicators of goodfit, we used a nonsignificant c χ2 test (p >.05), CFI, and TLI of .95 (or higher) and RMSEA point estimate of .06 (orlower) and upper confidence interval of .06 or (lower)  . Given a (lower) . All ana (lower) . The pubn = 67) of respondents were White, 34%(n = 67) Black or African American, and 32%(n = 64) Latinx or Hispanic. Overall, 78%(n = 155) of the sample, regardless of race/ethnicity,identified as gay, 14% (n = 28) as bisexual, and 8%(n = 15) as homosexual. Bivariate associations indicatedsignificant differences among White, Black, and Latinx participants on theircountry of birth and three of six SES indicators: employment, income, andfinances as a source of chronic strain. A higher proportion of Latinxrespondents  were born outside the United States,compared to White  and Black  respondents. A higher proportion of Whiterespondents reported being employed  compared totheir Black  and Latinx  peers. Similarly, a higher proportion of White gay and bisexual males  did not experience finances as a source of chronicstrain in the lives compared to Black  and Latinx(23% n = 15) gay and bisexual males. Last, 46%(n = 30) of White gay and bisexual males reported earningmore than $50,000 annually, compared to 16% (n = 10) and 28%(n = 19) of Latinx and Black gay and bisexual males,respectively.2  = 10.90, p =.28, RMSEA = .033, 90% CI , CFI = .987, TLI = .979). Standardizedparameter estimates are provided in p < .001) and greater than .40.The percentages of variance  in eachobserved item that is explained by the measurement model ranged from .22(chronic strain: residence) to .60 (employment). We did not conduct post-hocmodifications because of the adequate fit between the data and our measurementmodel.Results indicated good fit between our SES measurement model and observed data. We did not conduct post-hoc modifications because of the adequate fitbetween the data and the model.Our hypothesized structural model is described graphically in p =. 02).p = . 03)than their non-Black peers. Latinx males also had lower SES  compared to their non-Latinx peers.Additionally, being born in the United States  and increasing household size  were associated with lower SES.p < . 001)and with higher collective self-esteem at follow-up . The negative relationship betweenbaseline SES and internalized homophobia at follow-up was notstatistically significant .Baseline SES was negatively associated with everyday discrimination andinternalized homophobia and positively associated with collectiveself-efficacy, each assessed at 1-year follow-up. Higher SES at baselinewas significantly associated with less frequent experience of everydaydiscrimination at follow-up  and social  well-being. Internalizedhomophobia and everyday discrimination were negatively associated withboth measures of well-being. All associations were statisticallysignificant, except for the association between internalized homophobiaand social well-being. Greater internalized homophobia was associatedwith lower psychological  andsocial  well-being. More frequentexperience of everyday discrimination was associated with lowerpsychological  and social  well-being. All psychosocial andwell-being variables reflected follow-up assessments.Collective self-efficacy was positively and significantly associated withpsychological  and Latinx  males scored higher on the internalizedhomophobia scale at follow-up compared to their non-Black and non-Latinxcounterparts. Black (β = −0.087) and Latinx(β = −0.107) males reported lower follow-upcollective self-efficacy than their non-Black and non-Latinxcounterparts. The relationship between race/ethnicity and follow-upeveryday discrimination differed. Black (β = 0.004) males reported more,albeit marginal, everyday discrimination than their non-Blackcounterparts. Latinx  malesreported less everyday discrimination than their non-Latinxcounterparts. The relationship of race/ethnicity with internalizedhomophobia at follow-up was the only significant direct associationbetween race/ethnicity and psychosocial resources.p = .004). Higher SES was associated withhigher psychological well-being via collective self-efficacy , internalized homophobia , and everyday discrimination . Additionally, SES had a significant indirect effect on social well-being . Higher SES was associated with highersocial well-being via collective self-efficacy , internalized homophobia , and everyday discrimination . In both models, collective self-efficacyand everyday discrimination were statistically significant indirectpathways. For both measures of well-being, collective self-efficacy hadthe largest predictive validity, as illustrated by the magnitude of theregression coefficient.p = .06). Among Latinx gay and bisexual males,the indirect effect of baseline SES on follow-up everyday discriminationwas 0.211 (p = .01). Similarly, the indirect effect ofbaseline SES on collective self-efficacy at 1-year follow-up was −0.063(p = .07) for Black males and −0.135(p = .01) for Latinx males. Among Latinx males, theindirect effect of baseline SES on collective self-efficacy  and everyday discrimination , both measured at 1-year follow-up, waslarger than the direct effect of being Latinx on collectiveself-efficacy  and everydaydiscrimination . Also, none of thedirect associations of Latinx ethnicity with psychosocial factors wasstatistically significant. While race/ethnicity might not have asignificant direct association with psychosocial factors, SES is animportant third variable to examine. The addition of SES as a thirdvariable appears to explain the relationship, thus giving us a fullerpicture of how the relationship between race/ethnicity and psychosocialresources can be better understood and improved.p = .002) and statistically significant compared tothe direct effect of SES on social well-being . Another suppressor effect was illustratedby the indirect effect of being Latinx on collective self-efficacy asmediated by SES , which was largerand statistically significant compared to the direct effect of beingLatinx on collective self-efficacy . The third suppressor effect occurred when we examined theassociation of being Latinx and experience of everyday discrimination.While the direct association of Latinx and everyday discrimination wasnegative and not significant ,this relationship became positive and significant when SES was added asan indirect pathway . This type ofsuppressor effect is also called an incomplete mediation in which thedirect and mediated effects have the opposite signs (p =.743).Although most of the indirect relationships we evaluated illustratedmediation, three significant indirect associations are consideredsuppressor effects .Suppreste signs . In otheThe primary goal of this study was to examine the direct and indirect associations ofrace/ethnicity with psychosocial resources and SES with psychological and socialwell-being. Structural equation modeling results indicated that SES is an essentialdeterminant of psychosocial resources and psychological well-being for gay andbisexual men. Higher SES directly predicted higher collective self-efficacy, lowereveryday discrimination, and both higher psychological as well as social well-being1 year later. These results are in line with previous findings that indicate SES isa key determinant of health outcomes at the population level, and that higher SES isprotective against health problems associated with being a racial or sexual minority. For exaOur findings also suggest that race/ethnicity plays an important role beyond SES inpsychosocial resources. Black and Latinx gay and bisexual men reported higherinternalized homophobia compared to their non-Black and non-Latinx counterparts.Also, Black and Latinx men reported lower collective self-efficacy at follow-up.These findings align with previous studies that demonstrate poor health outcomesamong sexual minority people of color . OurstuOur results provide support for the role of race/ethnicity as well as SES ininfluencing psychosocial and social well-being. However, results also emphasize theneed for further research to understand the upstream determinants and mechanismsaccounting for the association between Black identity and internalized homophobia,as this was not mediated through SES. Considering the strong relationship betweenrace/ethnicity and SES, these results provide additional support for prospectivestudies and interventions in nationally representative and diverse populations. Ourstudy confirms that sexual minority people of color experience poorer psychologicalhealth outcomes, and these outcomes are mediated through SES. Our results may alsohave important implications for clinical trials and epidemiologic studies becausedifferent study endpoints may be differentially influenced by race/ethnicity. Ourstudy finds that SES indicators may account for the relationship betweenrace/ethnicity and psychosocial factors. This result is important given increasedresearch/findings indicating health disparities among people of color but does notfurther contextualize these findings in terms of socioeconomic differences amongracial/ethnic and sexual minorities.Critical to the interpretation of our findings is the recognition that SES is rootedin the sociopolitical history of race in the United States. In ouroperationalization of SES, we incorporated indicators, such as education,employment, chronic strain due to neighborhood conditions and net worth, that areconsidered as downstream consequences of institutionalized racism . The conA major strength of this study is the use of multiple SES indicators measured at theindividual level. We used education, income, employment status, net worth residence,and finances. Also, the study adopted a longitudinal design, assessing measures atbaseline and after a 1-year follow-up. Lastly, the study sample was diverse in termsof age, SES, and race/ethnicity, drawn from New York City neighborhoods. However,some important limitations in the current study should be noted. Methodologically,the self-reported nature of our data precludes drawing causal inferences from ourfindings because of recall or response biases. Also, mediation, as described in thecurrent study, was in the statistical sense only; replication with prospectivemeasurement of the mediators and outcomes would be needed to validate these models.Along these lines, the estimate of a third variable effect is subject to samplingvariability, and as a result, in any given sample, a variable may appear to act as amediator, confounder, or suppressor only due to chance . Also, tIn conclusion, our findings highlight the critical role of SES and race/ethnicity inthe psychological and social well-being of sexual minorities. These findingscorrespond with and extend intersectional frameworks, which have underscored sexualminority status and race/ethnicity as interactive, mutually constitutive factorsthat create various types of discrimination and oppression, which in turn, determineoutcomes and inequities in health. Specifically, this analysis brings attention toSES as an often under-conceptualized and invisible third variable that, alongsidesexual minority status and race/ethnicity, is crucial for intersectional frameworkson health. Therefore, interventions to address health inequities among sexualminorities must consider SES as an organizing concept that shapes people’s livedexperiences, exposure to stress, and resources for coping with stress. Moreover, thefindings reported here indicate the importance of psychological resources astargets for guiding psychosocial or public health interventions with theseintersectional populations. Given the observed differences between Black and Latinxgay and bisexual men, culturally informed approaches are necessary to consider howto enhance these psychological resources to improve the health of these distinctgroups.


Background: To comprehensively understand the impact of sex and smoking on the efficacy of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy in terms of overall survival (OS) in non-small-cell lung cancer (NSCLC).Methods: PubMed, Cochrane Library, Embase, and Scopus were searched from inception to March 17, 2019. OS was analyzed based on hazard ratios (HRs) and 95% confidence intervals (CIs) and estimated using the random effects model.Results: Our meta-analysis included 22 studies involving 11,874 patients. In the primary analysis, we found no statistically significant efficacy difference for EGFR-TKI intervention between females and males  and no obvious efficacy difference between never smokers and ever smokers . In the subgroup analysis of placebo control treatment, we found that female NSCLC patients who received EGFR-TKI therapy had a longer OS than male patients , while smoking status showed no significant effect on the efficacy of EGFR-TKI treatment in terms of the OS of NSCLC patients in all subgroup analyses.Conclusion: The efficacy of EGFR-TKI therapy for NSCLC patients is independent of smoking status but dependent on sex, and females have a longer OS than males. Lung cancer is one of the most common cancers in men and women, and there is no doubt that lung cancer poses the greatest threat to human life, as it results in one-quarter of all cancer deaths . HoweverOveractivation of epidermal growth factor receptor (EGFR) tyrosine kinases is a key mechanism leading to the development of NSCLC . In recePrevious meta-analyses focused on EGFR-mutated NSCLC patients and the progression-free survival (PFS) have concluded that female and non-smoking NSCLC patients have better efficacy with EGFR-TKIs than male patients and smokers –10. HoweIn the meantime, we found two meta-analyses that comprehensively and thoroughly studied the effect of sex on the efficacy in terms of OS of immune checkpoint inhibitors in cancer treatment , 12. We We performed this meta-analysis according to the PRISMA guidelines . PubMed,The literature inclusion criteria were as follows: randomized controlled clinical trials for NSCLC that contained any single EGFR-TKI treatment; the treatment plans in the corresponding control group did not contain any other EGFR-TKIs; the prognosis endpoint was OS, and the corresponding hazard ratios (HRs) and 95% confidence intervals (CIs) were reported according to sex and/or smoking status; and the full-text manuscripts were published in English. The exclusion criteria were as follows: retrospective studies of clinical cases; abstracts, reports, and papers from conferences; literature reviews and meta-analyses.Two authors independently extracted data from the included studies. Discrepancies were resolved by all authors through discussion to reach consensus. The following variables were extracted from each study: first author, publication year, EGFR mutation status, trial name, lines of therapy, EGFR-TKI intervention drug, control treatment plan, total number of patients, median age (years), median follow-up time (months), overall HR with 95% CI, HR with 95% CI according to patient sex, and HR with 95% CI according to smoking status. When duplicate publications were identified from one trial, we included only the most complete report.The methodological quality of the included studies was assessed using the five-point Jadad score , which jI2-values  smokers. For the overall HRs, we used the random effects model to calculate the pooled HR directly. For the HRs classified by sex and smoking status, we first calculated the interaction HRs and 95% CIs for each study and thereafter obtained the pooled HR using the random effects model. The heterogeneity between studies was identified using the Q-test and quantified using 2-values , 12. Pot , lines of therapy (>1 and 1), EGFR-TKI intervention , and control treatment .P-values are 2-sided, and a P-value of 0.05 indicated statistical significance. An HR < 1 indicated that EGFR-TKI efficacy was better than non-EGFR-TKI efficacy, EGFR-TKI efficacy in females was better than in males, and EGFR-TKI efficacy in never smokers was better than in ever smokers.We performed all data analyses using Stata 14.0 . All reported By searching our search terms in the database, we obtained 531 potential publications. A total of 136 records were excluded because of duplicated titles. By reviewing the abstract and full text, 375 records were further excluded according to our inclusion and exclusion criteria. Therefore, 20 publications were selected. In addition, by reviewing the references from these 20 selected studies, we found an additional 2 studies that were also in line with our inclusion criteria. Ultimately, 22 studies –36 were We assessed the quality of the 22 studies by the Jadad score. The mean score was 3.64 (ranging from 3 to 5), and no study received a low-quality score (scored 2 or less), indicating that these included studies possessed high methodological quality. The Jadad scores for each study are listed in the As shown in In particular, Ellis et al.  did not P = 0.05) in NSCLC (P = 0.30) (P = 0.31)  . Similar = 0.31) .P = 0.04) and smoking status interaction HRs  but not in sex interaction HRs   , 3. BothP = 0.04), while the other groups showed no statistically significant difference , lines of therapy (>1 and 1), EGFR-TKI intervention , and control treatment . According to the pooled interaction HRs of sex, we found a statistically significant OS advantage for females compared with males only for the EGFR-TKI intervention compared with the control placebo treatment (pooled HR 0.86, 95% CI 0.75–1.00, fference . FurtherOS and PFS are the main endpoints in clinical trials of cancer. It is well-known that PFS is not in line with OS in many cases,  and in sWe first demonstrated the advantage in OS for NSCLC patients who received EGFR-TKI intervention compared with other systemic therapies. Thereafter, we found no significant OS differences for EGFR-TKI intervention between the sexes and smoking status compared with other systemic therapies. Finally, in the subgroup analyses, when compared with placebo, we demonstrated that female NSCLC patients who received EGFR-TKI therapy had a longer OS than males. However, smoking status showed no significant effect on the efficacy of EGFR-TKI treatment in terms of the OS of NSCLC patients in all of our subgroup analyses.In recent years, significant sex-based differences in biology, epidemiology and treatment responses have become evident . There aIn a previous meta-analysis conducted by Lee et al. , EGFR-TKFor the studies on chemotherapy in patients with NSCLC, Wakelee et al.  reportedIt is well-known that tobacco smoking is an important cause of the development and progression of NSCLC. The incidence of EGFR mutations in NSCLC differs according to smoking history . EGFR muThe impact of smoking status on the efficacy of EGFR-TKIs in terms of PFS in NSCLC is contradictory according to previous meta-analyses , 10, 40.In summary, since sex and smoking status are the two main clinical features of lung cancer, our current research has important guiding significance for the clinical treatment of lung cancer. Our results suggest that we do not need to worry that smoking status will affect the efficacy of EGFR-TKIs and that EGFR-TKIs will have better efficacy in female patients than in male patients. However, on the other hand, the efficacy of EGFR-TKIs in male patients is not so ideal, indicating that more treatment options for male lung cancer patients need to be further developed in the future.Our current study has several limitations. First, as a meta-analysis, it relies on published results rather than the individual data of patients. Second, those excluded studies that lack published sex and smoking status subgroup analysis data may also contain potential differences. Finally, aside from sex and smoking status, differences in OS outcomes may be influenced by other non-pharmacological factors.Two main conclusions can be drawn from our current meta-analysis. The first is that the efficacy of EGFR-TKI therapy for NSCLC patients is sex-dependent, and females have a longer OS advantage than males. The second point is that smoking status has no effect on the efficacy of EGFR-TKI therapy in terms of the OS of NSCLC patients.All datasets generated for this study are included in the article/JX and QC: concept and design. JX and LZ: acquisition, analysis or interpretation of data, and statistical analysis. JX and BH: drafting of the manuscript. JX, BH, and QC: critical revision of the manuscript for important intellectual content. BH and QC: administrative, technical or material support, and study supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Although wheezing phenotypes have previously been accurately described using well-defined cohorts reporting longitudinal wheezing, early-life factors which lead to development of each wheezing phenotype remain uncertain . Birth weight and catch-up growth affect later respiratory outcomes [3], but the influence of weight gain on specific wheezing phenotypes in term-born children has not been described. Rapid weight gain in early-life is associated with increased rates of childhood wheeze and lower lung function . In one meta-analysis rapid infant weight gain was linked to pre-school wheeze and school-aged asthma; and to increased childhood respiratory symptoms in another meta-analysis . Effect of weight gain in early-life in term-born children on wheezing in early-life is less well reported [8]. In contrast, rapid increase in body mass index in infancy is associated with increased risk of asthma at school-age in preterm-born infants [9]. This study shows that catch-up growth in infancy is associated with increased early wheeze in childhood, so care is needed to not to excessively feed in early infancyhttps://bit.ly/2YPxtBw To the Editor:Although wheezing phenotypes have previously been accurately described using well-defined cohorts reporting longitudinal wheezing, early-life factors which lead to development of each wheezing phenotype remain uncertain , 2. BirtSince it is unclear if rapid weight gain in early-life affects any specific wheezing phenotype in term-born children, we investigated if catch-up growth in the first year of life was associated with any specific wheezing phenotypes.We confined our analyses to term-born (≥37 weeks' gestation), singleton, Caucasian children from the Millennium Cohort Study (MCS), who were born in the UK between 2000 and 2002 , 11. Datth and 20–80th centile for birth weight, respectively, corrected for gender and gestational age [e.g. family members, or formal, e.g. childminder or nursery.Birth weight and weight z-scores adjusted for gestational age and gender were calculated using the LMS Growth program  using the British 1990 reference values . Catch-uonal age . AntenatMultinomial logistic regression was performed and relative risk ratios and corresponding 95% confidence intervals for associations between catch-up growth in the first year and wheezing phenotypes (3 to 11 years) are reported. The “No wheezing” class was used as the reference group. PASW 23  was used to perform the analysis.Wheezing data were available for 9353 Caucasian, singleton, term-born children, with the complete dataset of interest available for 6161 children. The number of children included in the adjusted analysis decreased due to missing data as only children with complete data were included. As expected (since preterm-born children were excluded), the gestational age and birthweight of included participants were higher than for the excluded children. Rates of SES, delivery by CS and IUGR were lower, but breastfeeding was higher, in included participants compared to excluded participants.e.g. leptin) which may promote airway remodelling and hypersensitivity [e.g. maternal smoking, may be more important. The associations between antenatal smoking and later respiratory outcomes are well recognised. Antenatal smoking can lead to abnormal fetal lung development as well as abnormal lung function and increased rates of asthma and wheezing in childhood [These data show evidence that catch-up growth is associated with early wheeze, but not with persistent or late wheeze. As catch-up growth is generally promoted by clinicians for infants born with IUGR, it is important that the longer-term effect of catch-up growth is well defined and the risks and benefits clearly delineated. It seems that short-term catch-up growth could be detrimental leading to increased rates of early wheeze. This is possibly due to a lag in growth of the lungs, a process known as dysanapsis, whereby somatic growth exceeds that of lung growth. Moreover, there are potential effects of pro-inflammatory mediators released from excess adipose tissue (sitivity . Howeversitivity . In contsitivity , 5. We ohildhood . Lung vohildhood . This maIt would have been interesting to define IUGR using customised growth charts that take into account maternal factors associated with birthweight , as it iA strength of this study is that we used data from a large, well-characterised UK cohort reporting well-defined wheezing phenotypes from the age of 3 years. Data were collected accurately at face-to-face interviews. The same associations were reported using weighted, most-likely assigned wheezing phenotype methods and after adjustment for early-life factors. Limitations are loss of follow-up, a lack of complete data for all subjects, and the lack of lung function data, which was not collected. There was possibly overlap between the exposure to the early-life factor and wheezing occurring. As wheeze was first reported at 3 years, we were unable to remove this overlap. The MCS oversampled from areas of deprivation and ethnic minority groups. We only reported data for Caucasian children and adjusted for SES to try to limit the influence these factors had.In conclusion, we have reported the effect of catch-up growth in early-life and later wheezing phenotypes in term-born, singleton, Caucasian children. The results suggest that catch-up growth might have a detrimental effect on rates of early wheezing; thus, caution is required when instituting nutritional regimes to increase weight in infancy.10.1183/13993003.00884-2020.Shareable1This one-page PDF can be shared freely online.ERJ-00884-2020.ShareableShareable PDF


Over one-third of stroke patients has long-term cognitive impairment. The likelihood of cognitive dysfunction is poorly predicted by the location or size of the infarct. The macro-scale damage caused by ischaemic stroke is relatively localized, but the effects of stroke occur across the brain. Structural covariance networks represent voxelwise correlations in cortical morphometry. Atrophy and topographical changes within such distributed brain structural networks may contribute to cognitive decline after ischaemic stroke, but this has not been thoroughly investigated. We examined longitudinal changes in structural covariance networks in stroke patients and their relationship to domain-specific cognitive decline. Seventy-three patients  were scanned with high-resolution magnetic resonance imaging at sub-acute (3 months) and chronic (1 year) timepoints after ischaemic stroke. Patients underwent a number of neuropsychological tests, assessing five cognitive domains including attention, executive function, language, memory and visuospatial function at each timepoint. Individual-level structural covariance network scores were derived from the sub-acute grey-matter probabilistic maps or changes in grey-matter probability maps from sub-acute to chronic using data-driven partial least squares method seeding at major nodes in six canonical high-order cognitive brain networks . We then investigated co-varying patterns between structural covariance network scores within canonical distributed brain networks and domain-specific cognitive performance after ischaemic stroke, both cross-sectionally and longitudinally, using multivariate behavioural partial least squares correlation approach. We tested our models in an independent validation data set with matched imaging and behavioural testing and using split-half validation. We found that distributed degeneration in higher-order cognitive networks was associated with attention, executive function, language, memory and visuospatial function impairment in sub-acute stroke. From the sub-acute to the chronic timepoint, longitudinal structural co-varying patterns mirrored the baseline structural covariance networks, suggesting synchronized grey-matter volume decline occurred within established networks over time. The greatest changes, in terms of extent of distributed spatial co-varying patterns, were in the default mode and dorsal attention networks, whereas the rest were more focal. Importantly, faster degradation in these major cognitive structural covariance networks was associated with greater decline in attention, memory and language domains frequently impaired after stroke. Our findings suggest that sub-acute ischaemic stroke is associated with widespread degeneration of higher-order structural brain networks and degradation of these structural brain networks may contribute to longitudinal domain-specific cognitive dysfunction. Examining network-based degeneration, Veldsman and Cheng et al. show that higher-order structural covariance networks are associated with cognition in sub-acute stroke and that greater degeneration of these networks is associated with longitudinal cognitive decline after ischaemic stroke.  Cognitive decline is common after ischaemic stroke . InfarctBrain atrophy is a normal part of healthy aging, but it is accelerated in the presence of dementia causing pathologies  and cereWe examined structural covariance in our discovery data set in canonical higher-order cognitive brain networks in 73 stroke patients 3 months and 1 year after ischaemic stroke. We predicted that the integrity of SCNs at 3-month post-stroke would reflect cognitive performance. If longitudinal changes in SCNs were to be associated with longitudinal cognitive decline, the relationship at the sub-acute phase is unlikely to be epiphenomenal. Therefore, to further test our hypothesis, we investigated whether changes in SCNs from 3-month to 1-year post-stroke would be associated with changes in cognitive function. We tested our models in our validation data set, an independent data set with matched imaging and neuropsychological and cognitive testing and performed split-half analysis for longitudinal validation.Stroke patient data from the Cognition and Neocortical Volume after Stroke study  were incNeuropsychological and cognitive tests were administered at each timepoint, as outlined in the published protocol . BrieflyParticipants were scanned on a 3-Tesla Siemens Tim Trio scanner using a 12-channel head coil . The scan protocol comprised a high-resolution magnetization prepared rapid gradient recalled echo sequence , Trail-Making test (A) (TMT-A)  and Simple Reaction Time task  in the attention domain, the Trail-Making test (B) (TMT-B)  in the executive function domain, the Boston Naming Test (BNT)  and Controlled Oral Word Association Test (COWAT)-animals  in the language domain, the Hopkins Verbal Learning Test (HVLT)-Delay  in the memory domain and the Rey Complex Figure (RCF)-copy  in the visuospatial function domain (P < 0.001) and all 12 SCNs significantly contributed to this LV . Additional analyses controlling for GM/TIV or GM + WM/TIV again demonstrated similar findings  . The corn domain . These rn domain . Furthern domain . Finally this LV . The sigfindings .P < 0.001) and it explained 64.91% of variance of the PLS model. A positive correlation between the behavioural and the brain scores  was once again noted  in the attention domain, the controlled word association test-FAS in the language domain  and RCF-delay  in the memory domain (P < 0.001) accounting for between 41.15 and 60.10% of the variance in the PLS model across 10 split-half samples , We derived networks based on the covariance of the rate of change in GM volume from sub-acute to chronic stroke. Importantly, the regions showing significant co-variation with the seeds mirror those shown in the SCNs derived at the sub-acute timepoint . This coin noted . All SCNin noted . The cory domain . Highly y domain . In the  samples . The firCognitive decline is common after ischaemic stroke , 2014 anStructural covariance of the default mode, dorsal attention, executive control, salience, memory and language-related networks was associated with cognitive performance in the attention, executive function, language, memory and visuospatial domains, showing an association between topographical network organization in sub-acute stroke and cognitive performance. Structural covariance networks seeded from known network nodes replicated the topographical pattern of known functionally specific networks. Using a global ‘brain score’ and ‘behavioural score’ estimated from the PLS model, we found a significant correlation showing cognitive impairment associated with more damaged SCNs that we replicated in the independent validation data set. Structural covariance of major canonical networks is associated with cognitive performance in sub-acute stroke. Specifically, more damaged SCNs were related to deficits in attention, executive function, language, memory and visuospatial function. Attention was most implicated in this analysis with three tests of attention co-varying with the SCN LV, compared to a single test in other domains. This may reflect the frequency of attentional impairment seen in sub-acute stroke, or the overlap of attentional functions across other cognitive domains.As a further test of whether SCN integrity was associated with cognitive impairment after stroke, we examined covariance in the rate of longitudinal change in major brain networks and related this to changes in cognitive performance between sub-acute and chronic stroke. Covariance networks mirrored the SCNs derived at the sub-acute timepoint, suggesting that GM volume changes from the sub-acute to chronic phase were occurring within established, domain-specific networks. The greatest changes, in terms of extent of distributed spatial co-varying patterns, were in the default mode network and dorsal attention network.Degeneration of the major SCNs was associated with cognitive decline, specifically in the attention, memory and language domain. These results suggested that faster degradation of SCNs of bilateral dorsal attention, default mode, language-related networks as well as left dorsolateral prefrontal cortex, hippocampus and frontal insula was related to greater decline in attention, language and memory performance. These two findings are important, but should not be surprising, given that attentional deficits are a consistent feature of post-stroke cognitive impairment. Indeed, the most frequently impaired domains after ischaemic stroke are attention, memory and language . Up to 7What might be the mechanism resulting in widespread SCN changes, across all networks, after ischaemic stroke, even after accounting for age-related degeneration? Stroke may initiate or aggravate neurodegenerative processes above that seen in healthy aging . One plaThe study should be interpreted in light of its limitations. As it is often the case, the heterogeneity observed within stroke cohorts precludes detailed examination of different profiles of cognitive impairments as the sample size of each sub-group was too small for adequate statistical power. We controlled for infarct volume in the analysis, but did not take into account location, again the heterogeneity of the stroke types and infarct locations, make sub-group analyses under-powered. We conducted an independent validation analysis and confirmed our main findings, namely one significant LV accounting for most of the variance in the seed PLS models. Fairly equal contributions from all SCNs as well as significant correlations between brain and behavioural scores were observed in the sub-acute model, which is similar to the findings in the discovery analysis. However, there were some differences related to the SCN profiles and neuropsychological tests that correlated with the LV in the longitudinal model. This may be the result of the sample size used in the validation sample (one-third of the discovery sample). Alternatively, this might reflect a degree of dynamic change in cognition at this timepoint. Cognition is likely to stabilize as the time from the stroke increases. Future study will examine the longitudinal effects at even longer, likely even more stable timepoints collected in this protocol (up to 5 years). As a group, the median stroke severity (as measured by mRS and NIHSS) was relatively mild. Although this may limit generalizability of the finding to cohorts with more severe stroke, it also raises the possibility that SCNs and cognition maybe even more disrupted when stroke is not as mild as in this cohort. Finally, we carefully chose the seeds to derive our SCNs based on the existing literature. Emerging large functional network atlases . Future Cognitive decline after ischaemic stroke has been difficult to predict due to widespread effects of stroke on the brain. Using data-driven multivariate methods to examine cognition and canonical brain networks across cognitive domains, we show that structural covariance integrity of cognitive networks is associated with cognition at 3-month post-stroke and with longitudinal cognitive decline in attention, memory and language from sub-acute to chronic stroke. Structural covariance analyses of brain networks reveal widespread network disruptions associated with cognitive decline.fcaa155_Supplementary_DataClick here for additional data file.


To compare anti-vascular growth factor (anti-VEGF) pharmacotherapy with pan-retinal photocoagulation (PRP) for proliferative diabetic retinopathy (PDR).PubMed, Embase, Medline, the ClinicalTrials.gov and the Cochrane Central Register of Controlled Trials were reviewed systemically. Randomized controlled trials (RCT) on anti-VEGF therapy versus PRP or anti-VEGF agent combined with PRP versus PRP for PDR are eligible to be included. Outcome measures were regression and recurrence of neovascularization, change in best corrected vision acuity, development of vitreous hemorrhage, and need for vitrectomy. A meta-analysis was conducted using RevMan .Twelve RCTs with a total of 1026 eyes were identified. The meta-analysis results showed that regression of neovascularization did not vary significantly among different treatment regimens (P=0.06), whereas the recurrence of new vessels was significantly lower in PRP monotherapy (P < 0.00001). The best corrected visual acuity was significantly improved with anti-VEGF monotherapy or in the combined group than in the PRP groups . Odds ratio for post-treatment vitreous hemorrhage and vitrectomy rate between anti-VEGF therapy and PRP were 0.65 , and 0.24 .Our meta-analysis indicates that anti-VEGF pharmacotherapy is associated with superior visual acuity outcomes and less PDR-related complications. However, there is insufficient evidence to suggest anti-VEGF therapy as an alternative to PRP. Proliferative diabetic retinopathy (PDR) is the leading cause of blindness, especially among the population of working age. Neovascularization is the key feature of PDR, as vascular endothelial growth factor (VEGF) contributes significantly in the pathologic mechanism of PDR. Effective treatment option can facilitate better visual rehabilitation. Over the past four decades, pan-retinal photocoagulation (PRP) is still the mainstay treatment for PDR  in spiteWe conducted a literature search on PubMed, Embase, Medline, the ClinicalTrials.gov, and the Cochrane Central Register of Controlled Trials, from inception to October 2019. Randomized controlled trials were identified using the following key terms: proliferative diabetic retinopathy, anti-VEGF therapy, panretinal photocoagulation, bevacizumab/avastin, pegaptanib, ranibizumab, aflibercept. Reports of clinical trials were restricted to those making comparison between intravitreal anti-VEGF therapy with or without combination of PRP and PRP in PDR patients. Further, we manually searched the references of these original studies and reviewed potentially relevant articles to supplement the initial search.Real-world studies of anti-VEGF and PRP therapy for PDR published before search date were all included. The inclusion criteria to screen the captured publications in this meta-analysis were as follows: (1) randomized controlled trials; (2) patients with PDR 18 years and older, who were scheduled for intravitreal anti-VEGF therapy as an alternative or adjuvant treatment to PRP; (3) an observation period of at least 3 months after the treatment. Exclusion criteria were studies with additional interventions, and conditions like previous history of intraocular surgery, anticipated need for vitrectomy within 12 months, or other causes leading to retinal neovascularization. Meeting abstracts, full texts without any raw data for retrieval and review articles were also excluded. If multiple publications were based on the same population, we chose the trial with the largest number of patients.Two reviewers (SG and ZL) independently finished the electronic database search. Data were extracted, and methodological quality of these RCTs was assessed according to the Cochrane Collaboration's “Risk of Bias” tool from the Cochrane Handbook for Systematic Reviews of Intervention . Resultsbaseline − BCVAendpointPrimary outcome measures were anatomical outcomes, including complete regression and recurrence of neovascularization. Secondary outcome measure was the mean change in best corrected vision acuity (BCVA) from baseline to the end of follow-up period. If the mean change in BCVA was available from the original article, we use the data directly, otherwise, we calculated the data according to the following formula : BCVAR =Progression of post-treatment vitreous hemorrhage (PVH) and need for vitrectomy after different therapies were assessed as secondary outcome parameters as well.2 statistic  . Meta-antatistic , and regectively . A probaThe literature search process is summarized in The characteristics of twelve RCTs are summarized in 2 = 82%). Analysis of two studies comparing the combined therapy with PRP found that the odds ratio was 2.17 . There was no significant difference between any of these comparisons, indicating insufficient ability of anti-VEGF drugs to regress NV no matter as an alternative or adjunct to PRP.Complete regression rates of retinal neovascularization at the end of follow-up were obtained, and forest plot comparison is shown in One study reported the similar recurrence rates between anti-VEGF and PRP groups (P=0.62). Meta-analysis of the incidence of NV recurrence in Two studies listed BCVA outcomes using EDTRS at 36 and 52 weeks. Meta-analysis results in 2 = 38%). Detailed results could be seen in A total of 7 studies reported the incidence of PVH. The pooling results manifested that there was a significant difference between anti-VEGF therapy and PRP  with no heterogenicity, while combined therapy was not significantly different compared to PRP . Such findings were similar to PVH, which remind us of the clinical potential of anti-VEGF monotherapy for preventing post-treatment vitreous hemorrhage and vitrectomy.4 studies comparing anti-VEGF therapy with PRP were extractable. Forest plot comparison in Quality of Evidence from RCTs considering the safety and efficacy of anti-VEGF agents for PDR used to be low . CurrentAnti-VEGF therapy is believed to have promising neovascular regression outcomes in active PDR patients  though iIt cannot be neglected that weighing the relative benefits of PRP and anti-VEGF agents in PDR treatment is influenced by the presence of DME. Probability of vison-impairing DME development is lower in anti-VEGF group . BressleSeveral RCTs showed no significant improvement in visual acuity with the application of anti-VEGF agents . HoweverSecondary to PDR, vitreous hemorrhage (VH) can cause severe vision loss. A Cochrane Review  revealedPatients with multiple diabetes comorbidity, low compliance, and treatment fatigue are also the obstacles to overcome . Lin et Several limitations are unavoidable. First, the limited number of RCTs, especially high-quality large-scale RCTs, made our results less convincing. Second, the time points of these extracted data were inconsistent, varied from 3 months to 2 years. Many studies found that a large amount of PDR patients showed a recurrence of neovascularization beginning at 3 months , so we cIn conclusion, it is necessary to further assess the long-term visual, anatomical, and safety outcomes of anti-VEGF therapy. PRP is still irreplaceable in the field of PDR. Innovation of anti-VEGF agent delivery assume a vital role in the future. Our results may provide some insights into weighing the pros and cons among anti-VEGF agents, PRP and combined therapy in PDR treatment. Individualized treatment relying on patient's baseline condition, compliance level, and economic status is the most important part in promoting therapeutic effect and optimizing vision care. It remains to be elucidated whether anti-VEGF agent is a time-tested beneficial therapy like PRP.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.XS: study design, reviewed the manuscript. SG and ZL: database management and search strategies, meta-analysis, wrote, and reviewed the manuscript.Science and Technology Commission of Shanghai Municipality (No.18411964900), Shanghai Municipal Commission of Health and Family Planning (No.201740004) supported this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.