Project description:In Hawaii, a rapidly-evolving mutation in the field cricket Teleogryllus oceanicus silences males by interfering with the development of sound-producing structures on their forewings. The mutation is called flatwing (fw), and it persists because of natural selection imposed by an acoustically-orienting parasitoid. We examined gene expression differences between wild-type and mutant crickets, focusing on juvenile wing buds. We profiled mRNA expression levels using RNA-seq, and characterized the wing bud proteome using quantitative mass spectrometry. Accessing protein expression profiles under the same experimental conditions enabled us to test correspondence between the two ‘omic levels.
Project description:In Hawaii, a rapidly-evolving mutation in the field cricket Teleogryllus oceanicus silences males by interfering with the development of sound-producing structures on their forewings. The mutation is called flatwing (fw), and it persists because of natural selection imposed by an acoustically-orienting parasitoid. We examined gene expression differences between wild-type and mutant crickets, focusing on juvenile wing buds. We profiled mRNA expression levels using RNA-seq, and characterized the wing bud proteome using quantitative mass spectrometry.
Project description:Background Severe burn injury often leads to a state of immune paralysis, which markedly increases the risk of death from secondary bloodstream infection. However, the molecular mechanisms underlying post_x001E_burn immune paralysis, especially why it worsens infectious outcomes, remain poorly understood. This study aimed to elucidate the epigenetic regulatory mechanisms of post_x001E_burn immune paralysis and to evaluate the therapeutic potential of targeted intervention for secondary bloodstream infection. Methods A 30% total body surface area full_x001E_thickness (third_x001E_degree) burn model was established in C57BL/6J mice. Secondary bloodstream infection was induced by intravenous injection of Klebsiella pneumoniae or Pseudomonas aeruginosa on day 14 post_x001E_burn. Immune paralysis was assessed by monocyte phagocytosis, T_x001E_cell proliferation and IFN_x001E_γ secretion, and immune cell subset analysis. For mechanistic investigation, transcriptome sequencing was performed to identify key molecules, followed by bisulfite sequencing, molecular interaction assays, and in vivo CRISPRi/a_x001E_mediated knockdown or overexpression to delineate the epigenetic regulation and function. Finally, AAV_x001E_CRISPRi was used to knock down the target molecule in vivo, and its effects on immune paralysis and secondary infection outcomes were evaluated. Twenty_x001E_four severe burn patients and ten healthy volunteers were enrolled for clinical validation. Results Burn mice exhibited typical immune paralysis, characterized by decreased macrophage phagocytosis, impaired T_x001E_cell proliferation and IFN_x001E_γ secretion, increased M2/Treg ratios, and reduced pro_x001E_inflammatory cytokine levels. Mechanistically, PTPN6 was markedly up_x001E_regulated after burn and drove immune paralysis by suppressing the NF_x001E_κB and MAPK signaling pathways. Further investigation revealed that TET2 directly bound to the PTPN6 promoter and mediated its demethylation, leading to PTPN6 overexpression. In vivo knockdown of PTPN6 effectively alleviated immune paralysis, reduced bacterial load, attenuated multi_x001E_organ damage, and improved 14_x001E_day survival in burn mice (P < 0.01). Clinical sample analysis confirmed PTPN6 promoter demethylation, up_x001E_regulation of the TET2/PTPN6 axis, and immune dysfunction in peripheral blood of burn patients. Conclusions The key mechanism driving post_x001E_burn immune paralysis is “TET2_x001E_mediated PTPN6 promoter demethylation → PTPN6 up_x001E_regulation → suppression of NF_x001E_κB/MAPK pathways”. AAV_x001E_CRISPRi_x001E_mediated knockdown of PTPN6 effectively reverses immune paralysis and significantly improves the outcome of secondary bloodstream infection. This work provides a novel molecular basis for understanding post_x001E_burn immune dysfunction and identifies a potential therapeutic target for clinical intervention.
Project description:RNA interference (RNAi) functions as the major host antiviral defense in insects, while less is understood about how to utilize antiviral RNAi in controlling viral infection in insects. Enoxacin belongs to the family of synthetic antibacterial compounds based on a fluoroquinolone skeleton that has been previously found to enhance RNAi in mammalian cells. In this study, we showed that enoxacin efficiently inhibited viral replication of Drosophila C virus (DCV) and Cricket paralysis virus (CrPV) in cultured Drosophila cells. Enoxacin promoted the loading of Dicer-2-processed virus-derived siRNA into the RNA-induced silencing complex, thereby enhancing antiviral RNAi response in infected cells. Moreover, enoxacin treatment elicited an RNAi-dependent in vivo protective efficacy against DCV or CrPV challenge in adult fruit flies. In addition, enoxacin also inhibited replication of flaviviruses, including Dengue virus and Zika virus, in Aedes mosquito cells in an RNAi-dependent manner. Together, our findings demonstrated that enoxacin can enhance RNAi in insects, and enhancing RNAi by enoxacin is an effective antiviral strategy against diverse viruses in insects, which may be exploited as a broad-spectrum antiviral agent to control vector transmission of arboviruses or viral diseases in insect farming.
