The soil insect Bradysia odoriphaga (Diptera: Sciaridae) causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga) have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR). This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga.
As a model hemimetabolous insect species and an invasive urban pest that is globally distributed, the American cockroach, Periplaneta americana, is of great interest in both basic and applied research. Previous studies on P. americana neuropeptide identification have been based on biochemical isolation and molecular cloning. In the present study, an integrated approach of genomics- and peptidomics-based discovery was performed for neuropeptide identification in this insect species. First, 67 conserved neuropeptide or neurohormone precursor genes were predicted via an in silico analysis of the P. americana genome and transcriptome. Using a large-scale peptidomic analysis of peptide extracts from four different tissues (the central nervous system, corpora cardiac and corpora allata complex, midgut, and male accessory gland), 35 conserved (predicted) neuropeptides and a potential (novel) neuropeptide were then identified. Subsequent experiments revealed the tissue distribution, sex difference, and developmental patterns of two conserved neuropeptides (allatostatin B and short neuropeptide F) and a novel neuropeptide (PaOGS36577). Our study shows a comprehensive neuropeptidome and detailed spatiotemporal distribution patterns, providing a solid basis for future functional studies of neuropeptides in the American cockroach (data are available via ProteomeXchange with identifier PXD021660).
The effect of targeted expression of an anabolic isoform of basic fibroblast growth factor (FGF2) in osteoblastic lineage on tibial fracture healing was assessed in mice. Closed fracture of the tibiae was performed in Col3.6-18 kDaFgf2-IRES-GFPsaph mice in which a 3.6 kb fragment of type I collagen promoter (Col3.6) drives the expression of only the 18 kD isoform of FGF2 (18 kDaFgf2/LMW) with green fluorescent protein-sapphire (GFPsaph) as well as Vector mice (Col3.6-IRES-GFPsaph, Vector) that did not harbor the FGF2 transgene. Radiographic, micro-CT, DEXA, and histologic analysis of fracture healing of tibiae harvested at 3, 10 and 20 days showed a smaller fracture callus but accelerated fracture healing in LMWTg compared with Vector mice. At post fracture day 3, FGF receptor 3 and Sox 9 mRNA were significantly increased in LMWTg compared with Vector. Accelerated fracture healing was associated with higher FGF receptor 1, platelet derived growth factors B, C, and D, type X collagen, vascular endothelial cell growth factor, matrix metalloproteinase 9, tartrate resistant acid phosphatase, cathepsin K, runt-related transcription factor-2, Osterix and Osteocalcin and lower Sox9, and type II collagen expression at 10 days post fracture. We postulate that overexpression of LMW FGF2 accelerated the fracture healing process due to its effects on factors that are important in chondrocyte and osteoblast differentiation and vascular invasion.
High molecular weight FGF2 transgenic mice (HMWTg) phenocopy the Hyp mouse, homolog of human X-linked hypophosphatemic rickets with phosphate wasting and abnormal fibroblast growth factor (FGF23), fibroblast growth factor receptor (FGFR), Klotho and mitogen activated protein kinases (MAPK) signaling in kidney. In this study, we assessed whether short-term (24 h) in vivo administration of FGF23 neutralizing antibody (FGF23Ab) could rescue hypophosphatemia and impaired FGFR signaling in kidneys of HMWTg male mice. Bone mineral density and bone mineral content in 1-month-old HMWTg mice were significantly reduced compared with Control/VectorTg mice. Serum FGF23 was significantly increased in HMWTg compared with VectorTg. Serum phosphate was significantly reduced in HMWTg and was rescued by FGF23Ab. Serum parathyroid hormone (PTH) was significantly increased in HMWTg but was not reduced by FGF23Ab. 1, 25(OH) D was inappropriately normal in serum of HMWTg and was significantly increased in both Vector and HMWTg by FGF23Ab. Analysis of HMWTg kidneys revealed significantly increased mRNA expression of the FGF23 co-receptor Klotho, transcription factor mRNAs for early growth response-1 transcription factor (Egr-1), and c-fos were all significantly decreased by FGF23Ab. A significant reduction in the phosphate transporter Npt2a mRNA was also observed in HMWTg kidneys, which was increased by FGF23Ab. FGF23Ab reduced p-FGFR1, p-FGFR3, KLOTHO, p-ERK1/2, C-FOS, and increased NPT2A protein in HMWTg kidneys. We conclude that FGF23 blockade rescued hypophosphatemia by regulating FGF23/FGFR downstream signaling in HMWTg kidneys. Furthermore, HMWFGF2 isoforms regulate PTH expression independent of FGF23/FGFR signaling. J. Cell. Physiol. 232: 610-616, 2017. © 2016 Wiley Periodicals, Inc.
RuXian-I has traditionally been used as a remedy for breast hyperplasia in the Inner Mongolia Autonomous Region of China. As a first step toward the investigation of biomarkers associated with RuXian-I treatment, a proteome-wide analysis of rat breast tissue was conducted. First, rat breast hyperplasia was induced by injection of estradiol and progesterone. After treatment with RuXian-I, there is a marked decrease in the hyperplasia, as can be shown by decreases in the nipple diameter and the pathological changes in breast. Subsequently, we used an approach that integrates size-based 2D-DIGE, MALDI-TOF/TOF-MS, and bioinformatics to analyze data from the control group, the model group and the RuXian-I treatment group. Using this approach, seventeen affected proteins were identified. Among these, 15 (including annexin A1, annexin A2, superoxide dismutase [Mn], peroxiredoxin-1, translationally-controlled tumor protein and α B-crystallin) were significantly up-regulated in the model group and down-regulated upon treatment with RuXian-I, and two (Tpil protein and myosin-4) have the opposite change trend. The expression of annexin A1 was confirmed using immunohistochemistry. The expression of superoxide dismutase (SOD) activity was confirmed biochemically. These results indicated that RuXian-I treats rat breast hyperplasia through regulation of cell cycle, immune system, metabolic, signal transduction, etc. The differential expressions of these proteins (annexin A1, superoxide dismutase [Mn], alpha B-crystallins and translationally controlled tumor protein, among others) were associated with occurrence and metastasis of breast cancer. These findings might provide not only far-reaching valuable insights into the mechanism of RuXian-I action, but also leads for prognosis and diagnosis of breast hyperplasia and breast cancer.
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