Combined analysis of the proteome and metabolome provides insight into microRNA-1174 function in Aedes aegypti mosquitoes.

BACKGROUND: Pathogenic viruses can be transmitted by female Aedes aegypti (Ae. aegypti) mosquitoes during blood-meal acquisition from vertebrates. Silencing of mosquito- and midgut-specific microRNA (miRNA) 1174 (miR-1174) impairs blood intake and increases mortality. Determining the identity of the proteins and metabolites that respond to miR-1174 depletion will increase our understanding of the molecular mechanisms of this miRNA in controlling blood-feeding and nutrient metabolism of mosquitoes. METHODS: Antisense oligonucleotides (antagomirs [Ant]) Ant-1174 and Ant-Ct were injected into female Ae. aegypti mosquitoes at 12-20 h posteclosion, and depletion of miR-1174 was confirmed by reverse transcription quantitative real-time PCR (RT-qPCR). Ant-1174-injected and control mosquitoes were collected before the blood meal at 72 h post-injection for tandem mass tag-based proteomic analysis and liquid chromatography-tandom mass spectrometry non-target metabolomic analysis to identify differentially expressed proteins and metabolites, respectively. RNA interference (RNAi) using double-stranded RNA (dsRNA) injection was applied to investigate the biological roles of these differentially expressed genes. The RNAi effect was verified by RT-qPCR and western blotting assays. Triglyceride content and ATP levels were measured using the appropriate assay kits, following the manufacturers' instructions. Statistical analyses were conducted with GraphPad7 software using the Student's t-test. RESULTS: Upon depletion of mosquito- and midgut-specific miR-1174, a total of 383 differentially expressed proteins (DEPs) were identified, among which 258 were upregulated and 125 were downregulated. Functional analysis of these DEPs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment suggested that miR-1174 plays important regulatory roles in amino acid metabolism, nucleotide metabolism, fatty acid metabolism and sugar metabolism pathways. A total of 292 differential metabolites were identified, of which 141 were upregulated and 151 were downregulated. Integrative analysis showed that the associated differential proteins and metabolites were mainly enriched in a variety of metabolic pathways, including glycolysis, citrate cycle, oxidative phosphorylation and amino acid metabolism. Specifically, the gene of one upregulated protein in miR-1174-depleted mosquitoes, purine nucleoside phosphorylase (PNP; AAEL002269), was associated with the purine, pyrimidine and niacin-nicotinamide metabolism pathways. PNP knockdown seriously inhibited blood digestion and ovary development and increased adult mortality. Mechanically, PNP depletion led to a significant downregulation of the vitellogenin gene (Vg); in addition, some important genes in the ecdysone signaling and insulin-like peptide signaling pathways related to ovary development were affected. CONCLUSIONS: This study demonstrates differential accumulation of proteins and metabolites in miR-1174-depleted Ae. aegypti mosquitoes using proteomic and metabolomic techniques. The results provide functional evidence for the role of the upregulated gene PNP in gut physiological activities. Our findings highlight key molecular changes in miR-1174-depleted Ae. aegypti mosquitoes and thus provide a basis and novel insights for increased understanding of the molecular mechanism involved in a lineage-specific miRNA in mosquito vectors.

Identifying interactions of linked irrigated lake-groundwater system by combining hydrodynamic and hydrochemical method.

During the irrigation period, the interactions between the linked lake-groundwater systems are complicated and change. This is because natural and human activities are happening at the same time, which makes it harder to identify the interactions. This study uses data on water level, hydrochemistry, and hydrogen-oxygen stable isotopes to analyze the hydrodynamics, electrical conductivity (EC), isotopic characteristics, and spatial distribution of lake water and groundwater to reveal lake-groundwater interactions. The results indicate that the hydrochemical type of Chagan Lake and groundwater is dominated by the HCO3-Na type. The key hydrochemical indicator EC obtained by principal component analysis (PCA) can be used to reveal the lake-groundwater interaction, and the interaction should be identified by location according to the significant correlation between hierarchical clustering results and regional distribution. The lake body's geographic coefficient of variation for EC and δ18O is small, and irrigation return flow is one factor in the region's surface water's significant spatial variation for EC and δ18O. The three study methods indicate that the groundwater supplies the lake in the vicinity of the Huoling River-Hongzi Pool, while in other sections, the lake water leaks and replenishes the groundwater, exhibiting geographic inconsistency. The isotope method was employed as a support tool to determine that groundwater might recharge the lake at Xinmiao Pool. According to the calculations of the Mix SIAR model, the groundwater recharge contribution rate in the Xinmiao Pool section is approximately 51%, while in the remaining sections, the contribution rate of lake water to groundwater ranges from approximately 25% to 52%. Therefore, the identification of the interaction is crucial for the linked irrigated lake-groundwater system where water sources are scarce and threatened by agricultural pollution.

Knockout and Restoration Reveal Differential Functional Roles of PPARγ1 and PPARγ2 in Chicken Adipogenesis.

Peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of adipogenesis and is expressed as two isoforms, PPARγ1 and PPARγ2. Our previous lentiviral overexpression study showed that PPARγ1 and PPARγ2 differentially regulated proliferation, differentiation, and apoptosis of the immortalized chicken preadipocyte cell line (ICP2). However, we cannot rule out the possibility that the endogenous expression of PPARγ isoforms may compromise our findings. In this study, using the dual sgRNA-directed CRISPR/Cas9 system, we generated PPARγ (PPARγ-/-) and PPARγ2-specific knockout (PPARγ2-/-) ICP2 cell lines and investigated the differences in proliferation and differentiation among PPARγ-/-, PPARγ2-/-, and wild-type ICP2 cells. EdU proliferation assay showed that both PPARγ2-specific and PPARγ knockouts significantly increased the proliferation rates. Consistently, real-time RT-PCR analysis showed that both PPARγ2-specific and PPARγ knockouts significantly upregulated the expression of proliferation marker genes PCNA and cyclinD1. FACS analysis revealed that PPARγ knockout significantly increased the number of cells accumulating in the S phase and decreased the number of cells accumulating in the G1/G0 phase. Oil Red O staining and gene expression analysis showed both PPARγ2-specific and PPARγ knockouts dramatically reduced capacity for adipogenic differentiation. To corroborate our previous findings, PPARγ1 and PPARγ2 expression were restored in PPARγ-/- cells by using the lentiviruses expressing chicken PPARγ1 (LV-PPARγ1) and PPARγ2 (LV-PPARγ2), respectively. Subsequent assays showed that restoration of expression of either PPARγ1 or PPARγ2 suppressed proliferation and stimulated differentiation of the PPARγ-/- cells. By comparison, PPARγ2 had stronger anti-proliferative and pro-adipogenic effects than PPARγ1. To understand the molecular mechanism underlying their differential effects on differentiation of the PPARγ-/- cells, we performed RNA-seq in the PPARγ-/- cells in which individual PPARγ isoform expression was restored at 72 h of differentiation. Transcriptomic analysis revealed that restoring PPARγ1 expression caused far more differentially expressed genes (DEGs) than restoring PPARγ2 expression. GO and KEGG pathway enrichment analyses indicated that PPARγ1 and PPARγ2 had distinct and overlapping functions in adipogenesis. Taken together, our results clearly indicate that PPARγ1 and PPARγ2 differentially impact chicken adipogenesis.

Genomic organization, intragenic tandem duplication, and expression analysis of chicken TGFBR2 gene.

Transforming growth factor beta receptor Ⅱ (TGFBR2), a core member of the transforming growth factor-ß (TGF-ß) signaling pathway. To date, chicken TGFBR2 (cTGFBR2) genomic structure has not been fully explored. Here, the complete sequences of cTGFBR2 transcript isoforms were determined by 5' and 3' rapid amplification of cDNA ends (5' & 3' RACE) and reverse transcription polymerase chain reaction (RT-PCR); the tissue expression profiling of cTGFBR2 transcript isoforms was performed using quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that cTGFBR2 gene produced 3 transcript isoforms though alternative transcription initiation, splicing, and polyadenylation, which were designated as cTGFBR2-1, cTGFBR2-2, and cTGFBR2-3, respectively. These 3 cTGFBR2 transcript isoforms encoded 3 protein isoforms: cTGFBR2-1, cTGFBR2-2, and cTGFBR2-3. Duplication analysis revealed that, unlike other animal species, cTGFBR2 gene harbored a 5.5-kb intragenic tandem duplication. Tissue expression profiling in the 4-wk-old Arbor Acres (AA) broiler chickens showed that cTGFBR2-1 was ubiquitously expressed, with high expression in abdominal fat, subcutaneous fat, lung, gizzard, and muscle; cTGFBR2-2 was highly expressed in heart, kidney, gizzard, and muscle; cTGFBR2-3 was weakly expressed in all the tested chicken tissues. Tissue expression profiling in the 7-wk-old broiler chickens of the fat and lean lines of Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) showed that cTGFBR2-1 was significantly differentially expressed in all the tested tissues except heart, cTGFBR2-2 was significantly differentially expressed in all the tested tissues except subcutaneous fat and liver, and cTGFBR2-3 was significantly differentially expressed in all the tested tissues between the lean and fat lines. Intriguingly, in the fat line, the 3 cTGFBR2 transcript isoforms were expressed to varying degrees in all the 3 tested fat tissues, while in the lean line, only cTGFBR2-1 was expressed in all the 3 tested fat tissues. This is the first report of intragenic tandem duplication within TGFBR2 gene. Our findings pave the way for further studies on the functions and regulation of cTGFBR2 gene.