Misregulation of Alternative Splicing in a Mouse Model of Rett Syndrome

Mutations in the human MECP2 gene cause Rett syndrome (RTT), a severe neurodevelopmental disorder that predominantly affects girls. Despite decades of work, the molecular function of MeCP2 is not fully understood. Here we report a systematic identification of MeCP2-interacting proteins in the mouse brain. In addition to transcription regulators, we found that MeCP2 physically interacts with several modulators of RNA splicing, including LEDGF and DHX9. These interactions are disrupted by RTT causing mutations, suggesting that they may play a role in RTT pathogenesis. Consistent with the idea, deep RNA sequencing revealed misregulation of hundreds of splicing events in the cortex of Mecp2 knockout mice. To reveal the functional consequence of altered RNA splicing due to the loss of MeCP2, we focused on the regulation of the splicing of the flip/flop exon of Gria2 and other AMPAR genes. We found a significant splicing shift in the flip/flop exon toward the flop inclusion, leading to a faster decay in the AMPAR gated current and altered synaptic transmission. In summary, our study identified direct physical interaction between MeCP2 and splicing factors, a novel MeCP2 target gene, and established functional connection between a specific RNA splicing change and synaptic phenotypes in RTT mice. These results not only help our understanding of the molecular function of MeCP2, but also reveal potential drug targets for future therapies

iTRAQ-based comparative proteomic analysis of cells infected with Eimeria tenella sporozoites

Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. When E. tenella infects a host cell, the host produces a corresponding change to deal with damage caused by this infection. To date, our knowledge on the mechanism of how the host cell responds to E. tenella infection is highly limited at both the molecular and cellular levels. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS was used to screen the differentially expressed proteins (DEPs) in BHK-21 cells infected with E. tenella sporozoites for 24 h post infection. In total, 6139 non-redundant distinct proteins were identified and 195 of these were found to have a fold change ratio ≥1.3 or ≤0.7 and p < 0.05, including 151 up-regulated proteins and 44 down-regulated proteins. The reliability of the proteomic data was further validated with qPCR and western blot. Gene Ontology enrichment indicated that the up-regulated DEPs were mainly involved in binding and catalytic activity, whereas the down-regulated DEPs were catalytic activity and molecular function regulators. Furthermore, KEGG pathway analysis showed that the DEPs participated in the PI3K-Akt, chemokine, Ras, Wnt, and p53 signaling pathways and so on, and the up-regulated and down-regulated DEPs mainly related to the ribosome and mRNA surveillance pathway, respectively. The data in this study provide an important basis to further analyze E. tenella host cell interactions

The role of perceived effectiveness of policy measures in predicting recycling behaviour in Hong Kong

Hong Kong is a compact and ‘land-hungry’ city where recycling has become an important measure for treating waste and reducing the demand on the limited landfills. The objectives of this paper are to extend the model of recycling attitude and behaviour to explain the relationship between perceived policy effectiveness and recycling behaviour. Previous studies on recycling attitude and behaviour had widely adopted the Theory of Planned Behaviour (TPB) and the Norm Activation Model (NAM), and drawn policy implications from them. Nevertheless, little research has been conducted to investigate the role of perceived effectiveness of policy measures in predicting recycling behaviour. To address this, a model that integrates the TPB and NAM was proposed in this study, and a street survey was conducted to investigate the case. The results illustrate that recycling intention is influenced by subjective norms, perceived behavioural control, moral norms, and awareness of consequences, as well as a newly proposed construct, namely perceived policy effectiveness. The study proved self-reported recycling behaviour (direct behaviour) and support for policy measures (indirect behaviour) are influenced by recycling intention. All in all the Government should provide more recycling bins, guidelines on recycling activities, and should accent what it has been doing to facilitate and encourage recycling

The checklist of protozoan and acanthocephalan from frogs in China

In order to understand the species of protozoan and acanthocephalan from frogs and their geographic distributions in China.Relevant literatures that reported protozoan and acanthocephalan in frogs of China were collected and a checklist was provided according to newer classification system on protozoan and acanthocephalan based on these literatures.In summary,61 species of protozoan and 8 species of acanthocephalan have been recorded in 31 species of frogs in China.Among them,the species of protozoan belong to 7 phyla,11 classes,12 orders,14 families,21 genera, and the species of acanthocephalan belong to 1 phylum,2 classes,2 orders,2 families,3 genera.The Chinese and Latin names,the namer and naming years,the parasitic hosts and parts,the geographical distributions and the sources of literatures of these protozoan and acanthocephalan species were listed as well,which provids the basic materials for a more comprehensive understand about species of protozoan and acanthocephalan from frogs in China

Identification and characterization of Eimeria tenella apical membrane antigen-1 (AMA1).

Apical membrane antigen-1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. In this study, a full-length cDNA of AMA1 was identified from Eimeria tenella (Et) using expressed sequence tag and the rapid amplification of cDNA ends technique. EtAMA1 had an open reading frame of 1608 bp encoding a protein of 535 amino acids. Quantitative real-time PCR analysis revealed that EtAMA1 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second-generation merozoites). The ectodomain sequence was expressed as recombinant EtAMA1 (rEtAMA1) and rabbit polyclonal antibodies raised against the rEtAMA1 recognized a 58-kDa native parasite protein by Western Blotting and had a potent inhibitory effect on parasite invasion, decreasing it by approximately 70%. Immunofluorescence analysis and immunohistochemistry analysis showed EtAMA1 might play an important role in sporozoite invasion and development

Molecular characterization and analysis of drug resistance-associated protein enolase 2 of Eimeria tenella

Eimeria tenella, an intestinal parasite, has brought huge economic losses to the poultry industry. The prevalence and severity of the development of drug resistance has increased the challenge of coccidiosis control. We previously identified the enolase 2 of E. tenella (EtENO2) was differentially expressed in drug-sensitive (DS) and drug-resistant strains using RNA-seq. In this study, the expression of EtENO2 in diclazuril-resistant (DZR), maduramicin-resistant (MRR), and salinomycin-resistant (SMR) strains was analyzed by quantitative real-time PCR (qRT-PCR) and western blots. EtENO2 was highly expressed in several drug-resistant strains compared with the DS strain. The qRT-PCR showed that the transcription level of EtENO2 in the field-isolated resistant strains was upregulated compared with the DS strain. The enzyme activity results indicated that the catalytic activity of EtENO2 in the drug-resistant strains was higher than in the DS strain. In addition, qRT-PCR and western blots showed that the expression level of EtENO2 was higher in second generation merozoites (SM) and unsporulated oocysts (UO) than that in sporozoites (SZ) and sporulated oocysts (SO). Immunofluorescence localization revealed that EtENO2 was distributed throughout SZ and SM and on the surface of the parasites. After the SZ invasion DF-1 cells, it was also observed on the parasitophorous vacuole membrane. Our secretion experiments found that EtENO2 could be secreted outside the SZ. This study indicated that EtENO2 might be related to the interaction between E. tenella and host cells and be involved in the development of E. tenella resistance to some anticoccidial drugs

Identification of an interaction between calcium-dependent protein kinase 4 (EtCDPK4) and serine protease inhibitor (EtSerpin) in Eimeria tenella

Abstract Background Eimeria tenella is an obligate intracellular apicomplexan protozoan parasite that has a complex life-cycle. Calcium ions, through various calcium-dependent protein kinases (CDPKs), regulate key events in parasite growth and development, including protein secretion, movement, differentiation, and invasion of and escape from host cells. In this study, we identified proteins that interact with EtCDPK4 to lay a foundation for clarifying the role of CDPKs in calcium channels. Methods Eimeria tenella merozoites were collected to construct a yeast two-hybrid (Y2H) cDNA library. The Y2H system was used to identify proteins that interact with EtCDPK4. One of interacting proteins was confirmed using bimolecular fluorescence complementation and co-immunoprecipitation in vivo. Co-localization of proteins was performed using immunofluorescence assays. Results Eight proteins that interact with EtCDPK4 were identified using the Y2H system. One of the proteins, E. tenella serine protease inhibitor 1 (EtSerpin), was further confirmed. Conclusion In this study, we screened for proteins that interact with EtCDPK4. An interaction between EtSerpin and EtCDPK4 was identified that may contribute to the invasion and development of E. tenella in host cells