Transcriptome Analysis Reveals Candidate Genes Involved in Light-Induced Primordium Differentiation in Pleurotus eryngii

Pleurotus eryngii, a highly valued edible fungus, is one of the major commercially cultivated mushrooms in China. The development of P. eryngii, especially during the stage of primordium differentiation, is easily affected by light. However, the molecular mechanism underlying the response of primordium differentiation to light remains unknown. In the present study, primordium expression profiles under blue-light stimulation, red-light stimulation, and exposure to darkness were compared using high-throughput sequencing. A total of 16,321 differentially expressed genes (DEGs) were identified from three comparisons. GO enrichment analysis showed that a large number of DEGs were related to light stimulation and amino acid biosynthesis. KEGG analyses demonstrated that the MAPK signaling pathway, oxidative phosphorylation pathway, and RNA transport were most active during primordium differentiation. Furthermore, it was predicted that the blue-light photoreceptor WC-1 and Deoxyribodipyrimidine photolyase PHR play important roles in the primordium differentiation of P. eryngii. Taken together, the results of this study provide a speculative mechanism that light induces primordium differentiation and a foundation for further research on fruiting body development in P. eryngii.

Integration of whole genome sequencing and transcriptomics reveals a complex picture of the reestablishment of insecticide resistance in the major malaria vector Anopheles coluzzii

Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids, and a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance.

Dynamics of Energy Metabolism in Carbon Starvation-Induced Fruitlet Abscission in Litchi

Fruit abscission is triggered by multiple changes in endogenous components of the fruit, including energy metabolism. However, it is still unknown how the core energy metabolism pathways are modified during fruit abscission. Here, we investigated the relationship between carbon starvation-induced fruitlet abscission and energy metabolism changes in litchi. The fruitlet abscission of litchi ‘Feizixiao’ was induced sharply by girdling plus defoliation (GPD), a carbon stress treatment. Using liquid chromatography tandem mass spectrometry (LC-MS/MS) targeted metabolomics analysis, we identified a total of 21 metabolites involved in glycolysis, TCA cycle and oxidative phosphorylation pathways. Among them, the content of most metabolites in glycolysis pathways and TCA cycles was reduced, and the activity of corresponding metabolic enzymes such as ATP-dependent phosphofructokinase (ATP-PFK), pyruvate kinase (PK), citrate synthase (CS), succinate thiokinase (SAT), and NAD-dependent malate dehydrogenase (NAD-MDH) was decreased. Consistently, we further showed that the expression of the relative genes (LcPFK2, LcPK2, LcPK4, LcCS1, LcCS2, LcSAT, LcMDH1 and LcMDH2) was also significantly down-regulated. In contrast, the level of ATP, an important metabolite in the oxidative phosphorylation pathway, was elevated in parallel with both higher activity of H+-ATPase and the increased expression level of LcH+-ATPase1. In conclusion, our findings suggest that carbon starvation can induce fruitlet abscission in litchi probably by energy depletion that mediated through both the suppression of the glycolysis pathway and TCA cycle and the enhancement of the oxidative phosphorylation pathway.

CSIG-27. PROTEOMIC ANALYSIS OF GENETICALLY STRATIFIED MENINGIOMA

WHO grade II and III as well as some WHO grade I meningioma are clinically aggressive. Approximately 60% sporadic meningiomas harbour mutations in the NF2 gene, others in genes including TRAF7, KLF4, AKT1, SMO and PIK3CA . However, the molecular mechanisms behind meningioma tumourigenesis are still unclear. We aim to identify novel biomarkers and therapeutic targets of meningioma by characterising the proteomic landscape. We performed (phosho)proteomic profiling of grade I, II and III meningiomas and three different mutational groups: AKT1 E17K /TRAF7, KLF4 K409Q /TRAF7 and NF2 -/-. We validated differential expression of proteins and phosphoproteins by Western blot on a meningioma validation set and by immunohistochemistry. Looking at all grades bioinformatics analysis revealed commonly upregulated proteins and phosphoproteins to be enriched in Gene Ontology terms associated with RNA metabolism. Validation studies confirmed significant overexpression of proteins such as EGFR and CKAP4 and upregulation and activation of the NIMA-related kinase, NEK9, involved in mitotic progression. Novel proteins described included the nuclear proto-oncogene SET, the splicing factor SF2/ASF and the higher-grade specific protein, Hexokinase 2. For the mutation subtypes we have quantified 4162 proteins across all mutational meningioma subgroups with proteomic profiles of mutational subgroups. Comparative analysis showed 10 proteins were commonly significantly upregulated among all mutational subtypes vs. normal meninges, indicating proteomic landscapes of mutational subtypes to be highly variable. 257 proteins were commonly significantly downregulated and enriched with molecular functions including aldehyde dehydrogenase and oxido-reductase. Mutational subtype-specific analysis identified 162 proteins significantly upregulated in AKT1 E17K /TRAF7 vs. remaining sample groups to be enriched in the oxidative phosphorylation pathway. Less proteins were commonly significantly upregulated in KLF4 K409Q /TRAF7 and NF2 -/- mutant meningioma subtypes respectively. Several of these up-regulated proteins including ANNEXIN-3, CRABP2, CLIC3 were verified. Analyses of 6600 phospho-sites predicted regulatory kinases. Further validation and functional verification of potential candidates is ongoing.

PhosSNPs-Regulated Gene Network and Pathway Significant for Rheumatoid Arthritis

<b><i>Objectives:</i></b> Peripheral blood mononuclear cells (PBMCs) are critical for immunity and participate in multiple human diseases, including rheumatoid arthritis (RA). PhosSNPs are nonsynonymous SNPs influencing protein phosphorylation, thus probably modulate cell signaling and gene expression. We aimed to identify phosSNPs-regulated gene network/pathway potentially significant for RA. <b><i>Methods:</i></b> We collected genome-wide phosSNP genotyping data and transcriptome-wide mRNA expression data from PBMCs of a Chinese sample. We discovered and verified with public datasets differentially expressed genes (DEGs) associated with RA, and replicated RA-associated SNPs in our study sample. We performed a targeted expression quantitative trait locus (eQTL) study on significant phosSNPs and DEGs. <b><i>Results:</i></b> We identified 29 nominally significant eQTL phosSNPs and 83 target genes, and constructed comprehensive regulatory/interaction networks, highlighting the vital effects of two eQTL phosSNPs (rs371513 and rs4824675, FDR &#x3c;0.05) and four critical node genes (HSPA4, NDUFA2, MRPL15, and ATP5O). Besides, two node/key genes NDUFA2 and ATP5O, regulated by rs371513, were significantly enriched in mitochondrial oxidative phosphorylation pathway. Besides, four pairs of eQTL effects were replicated independently in whole blood and/or transformed fibroblasts. <b><i>Conclusions:</i></b> The findings delineated a potential role of protein phosphorylation and genetic variations in RA and warranted the significant roles of phosSNPs in regulating RA-associated genes expression in PBMCs. The results pointed out the relevance and significance of oxidative phosphorylation pathway to RA.

OS08.5.A Proteomic analysis of meningioma

BACKGROUND Meningioma is the most common primary intracranial tumor. Although ~80% are benign some WHO grade I are clinically aggressive. Chemotherapies are ineffective and biomarkers for clinical management are lacking. Approximately 60% sporadic meningiomas harbor mutations in the NF2 gene andutations in TRAF7, KLF4, AKT1, SMO and PIK3CA have been identified in the majority NF2-positive tumors esp lower grade. However, the molecular mechanisms behind meningioma tumourigenesis is still unclear. We aim to identify novel biomarkers and therapeutic targets of meningioma by characterizing the proteomic landscape. MATERIAL AND METHODS We analysed grade I, II and III frozen meningioma specimens and three different mutational groups: AKT1/TRAF7, KLF4/TRAF7 and NF2 -/- using LC-MS/MS to analyse global proteins, enriched phosphoproteins and phosphopeptides. Differential expression and functional annotation of proteins was completed using Perseus, IPA® and DAVID. For mutational subtypes quantitative phosphoproteomics was performed using TMT 10plex labeling approach followed by motif analysis using motif-X algorithm. We validated differential expression of proteins and phosphoproteins by Western blot and immunohistochemistry. RESULTS We quantified 3888 proteins and 3074 phosphoproteins across all meningioma grades. Bioinformatics analysis revealed commonly upregulated (phospho)proteins to be enriched in Gene Ontology terms associated with RNA metabolism. Validation confirmed significant overexpression of proteins such as EGFR, CKAP4, the nuclear proto-oncogene SET, the splicing factor SF2/ASF as well as total and activated phosphorylated form of the NIMA-related kinase, NEK9, involved in mitotic progression. Hexokinase 2 was overexpressed in higher grades. For the mutation subtypes we have quantified 4162 proteins across all mutational meningioma subgroups. Analysis showed distinct proteomic profiles of mutational subgroups. Comparative analysis showed 10 proteins were commonly significantly upregulated among all mutational subtypes vs. normal meninges. 257 proteins were commonly significantly downregulated and enriched with molecular functions including aldehyde dehydrogenase and oxido-reductase. Mutational subtype-specific analysis identified 162 proteins significantly upregulated in AKT1/TRAF7 vs. remaining sample groups to be enriched in the oxidative phosphorylation pathway. 14 and 7 proteins were commonly significantly upregulated in KLF4/TRAF7 and NF2 -/- mutant meningioma subtypes respectively. Several of these up-regulated proteins including ANNEXIN-3, CRABP2, CLIC3 and Endoglin were verified via WB. Lastly, analyses of 6600 phosphosites predicted regulatory kinases CONCLUSION We show extensive proteomic and phospophoproteomics analysis of meningioma and suggest new therapeutic and biomarker candidates.

Integration of whole genome sequencing and transcriptomics reveals a complex picture of insecticide resistance in the major malaria vector Anopheles coluzzii

Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An. coluzzii: a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance.

Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid β-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal β-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid β-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid β-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.

Oxidative Phosphorylation Is Dysregulated Within the Basocortical Circuit in a 6-month old Mouse Model of Down Syndrome and Alzheimer’s Disease

Down syndrome (DS) is the primary genetic cause of intellectual disability (ID), which is due to the triplication of human chromosome 21 (HSA21). In addition to ID, HSA21 trisomy results in a number of neurological and physiological pathologies in individuals with DS, including progressive cognitive dysfunction and learning and memory deficits which worsen with age. Further exacerbating neurological dysfunction associated with DS is the concomitant basal forebrain cholinergic neuron (BFCN) degeneration and onset of Alzheimer’s disease (AD) pathology in early mid-life. Recent single population RNA sequencing (RNA-seq) analysis in the Ts65Dn mouse model of DS, specifically the medial septal cholinergic neurons of the basal forebrain (BF), revealed the mitochondrial oxidative phosphorylation pathway was significantly impacted, with a large subset of genes within this pathway being downregulated. We further queried oxidative phosphorylation pathway dysregulation in Ts65Dn mice by examining genes and encoded proteins within brain regions comprising the basocortical system at the start of BFCN degeneration (6 months of age). In select Ts65Dn mice we demonstrate significant deficits in gene and/or encoded protein levels of Complex I-V of the mitochondrial oxidative phosphorylation pathway in the BF. In the frontal cortex (Fr Ctx) these complexes had concomitant alterations in select gene expression but not of the proteins queried from Complex I-V, suggesting that defects at this time point in the BF are more severe and occur prior to cortical dysfunction within the basocortical circuit. We propose dysregulation within mitochondrial oxidative phosphorylation complexes is an early marker of cognitive decline onset and specifically linked to BFCN degeneration that may propagate pathology throughout cortical memory and executive function circuits in DS and AD.