Research Note: Preliminary functional analysis of EGF-like domains of Eimeria tenella microneme protein 7 identified in sporozoites and merozoites.

Eimeria tenella, an obligate intracellular apicomplexan parasite, is the major causative agent of chicken coccidiosis. Some epidermal growth factor (EGF)-like domain-containing proteins of other members of apicomplexan parasites have been reported to contribute to parasite survival. To date, however, EGF-like domain-containing proteins of E. tenella are not well studied. In this study, a gene fragment that encodes 4 EGF-like domains of E. tenella microneme protein 7 (EGF-EtMIC7) was amplified and expressed using an Escherichia coli expression system. Following generation of polyclonal antibodies that recognize recombinant EGF-EtMIC7 (rEGF-EtMIC7), the expression of EtMIC7 in sporozoites and merozoites was examined. Moreover, its roles in cellular regulation were investigated. The native EtMIC7 in E. tenella sporozoites and merozoites was detected by using Western blot and indirect immunofluorescence assays. rEGF-EtMIC7 could activate Akt, whereas blockade of EGF receptor (EGFR) failed to induce Akt phosphorylation. Compared with the control group, LMH cells treated with rEGF-EtMIC7 showed increased cell proliferation and expressed higher levels of B cell leukemia/lymphoma 2 (BCL-2). These findings contribute to the better understanding of parasite-host interactions at the molecular level during E. tenella infection.

Research Note: Transcriptomic analysis of LMH cells in response to the overexpression of a hypothetical protein identified in Eimeria tenella SD-01 strain.

Though genome sequencing of Eimeria tenella predicts more than 8,000 genes, the molecular functions of many proteins remain unknown. In this study, the coding region corresponding to the mature peptide of a hypothetical protein of E. tenella (ETH_00023950) was amplified and expressed in a bacterial system. Following preparation of polyclonal antibody that recognizes ETH_00023950, the expression of ETH_00023950 in merozoites was examined. Meanwhile, we determined the transcriptomic responses of the leghorn male hepatoma (LMH) cells to its expression. Sequencing analysis showed that one single nucleotide polymorphism and one indel of ETH_00023950 of E. tenella SD-01 strain were found compared with that of the UK reference Houghton strain, leading to a frame shift and a premature stop codon. The expression of ETH_00023950 in E. tenella merozoites was confirmed by indirect immunofluorescence and Western blot analysis. Transcriptomic analysis showed that ETH_00023950 altered the expression of 2,680 genes (321 downregulated genes and 2,359 upregulated genes) in LMH cells. The RNA-sequencing data were consistent with the results of the quantitative real-time polymerase chain reaction (qRT-PCR). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that differentially expressed transcripts were significantly related to 8 pathways, including oxidative phosphorylation and TGF-beta signaling pathway. These findings contribute to understanding host-pathogen interaction and secondary bacterial infections related to E. tenella.

Transcriptional changes in LMH cells induced by Eimeria tenella rhoptry kinase family protein 17.

Though a number of Eimeria tenella rhoptry kinase family proteins have been identified, little is known about their molecular functions. In the present study, the gene fragment encoding the matured peptide of E. tenella rhoptry kinase family protein 17 (EtROP17) was used to construct a recombinant vector, followed by transfection into leghorn male hepatoma (LMH) cells. Then, the transcriptional changes in the transfected cells were determined by RNA-seq. The expression of EtROP17 in LMH cells was validated by both Western blot and indirect immunofluorescence analysis. Our analysis showed that EtROP17 altered the expression of 309 genes (114 downregulated genes and 195 upregulated genes) in LMH cells. The quantitative real-time polymerase chain reaction (qRT-PCR) results of the selected differentially expressed genes (DEGs) were consistent with the RNA-seq data. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were significantly enriched in nine pathways, such as toll-like receptor signaling pathway, ECM-receptor interaction, intestinal immune network for IgA production and focal adhesion. These findings reveal several potential roles of EtROP17, which contribute to understanding the molecular mechanisms underlying the host-parasite interplay.

Identification and Protective Efficacy of Eimeria tenella Rhoptry Kinase Family Protein 17.

Eimeria tenella encodes a genome of approximately 8000 genes. To date, however, very few data are available regarding E. tenella rhoptry kinase family proteins. In the present study, the gene fragment encoding the mature peptide of the rhoptry kinase family protein 17 of E. tenella (EtROP17) was amplified by PCR and expressed in E. coli. Then, we generated polyclonal antibodies that recognize EtROP17 and investigated the expression of EtROP17 in the merozoite stage of E. tenella by immunofluorescent staining and Western blot analysis. Meanwhile, the protective efficacy of rEtROP17 against E. tenella was evaluated in chickens. Sequencing analysis showed that a single base difference at sequence position 1901 was observed between the SD-01 strain and the Houghton strain. EtROP17 was expressed in the merozoite stage of E. tenella. The results of the animal challenge experiments demonstrated that vaccination with rEtROP17 significantly reduced cecal lesions and oocyst outputs compared with the challenged control group. Our findings indicate that EtROP17 could serve as a potential candidate for developing a new vaccine against E. tenella.

A novel free-standing metal organic frameworks-derived cobalt sulfide polyhedron array for shuttle effect suppressive lithium-sulfur batteries.

Metal-organic-frameworks-derived nanostructures have received broad attention for secondary batteries. However, many strategies focus on the preparation of dispersive materials, which need complicated steps and some additives for making electrodes of batteries. Here, we develop a novel free-standing Co9S8polyhedron array derived from ZIF-67, which grows on a three-dimensional carbon cloth for lithium-sulfur (Li-S) battery. The polar Co9S8provides strong chemical binding to immobilize polysulfides, which enables efficiently suppressing of the shuttle effect. The free-standing S@Co9S8polyhedron array-based cathode exhibits ultrahigh capacity of 1079 mAh g-1after cycling 100 times at 0.1 C, and long cycling life of 500 cycles at 1 C, recoverable rate-performance and good temperature tolerance. Furthermore, the adsorption energies towards polysulfides are investigated by using density functional theory calculations, which display a strong binding with polysulfides.

Comparison of radius of anterior lens surface curvature measurements in vivo using the anterior segment optical coherence tomography and Scheimpflug imaging.

BACKGROUND: To assess the radius of anterior lens surface curvature (RAL) measurements with anterior segment optical coherence tomography (AS-OCT) in comparison with Scheimpflug imaging. METHODS: This prospective, cross-sectional study was carried out at Zhongshan Ophthalmic Center, Guangzhou, China. We enrolled 59 eyes, including 30 eyes from 30 cataractous volunteers (59 to 87 years) and 29 eyes from 29 young participants (19 to 49 years). After mydriasis, the RAL was measured automatically by the built-in software in the AS-OCT (CASIA 2). The Scheimpflug images were measured with the build-in caliper tool of the Scheimpflug camera (Pentacam), and RAL were further calculated with the principle of best-fitted circle. Intraobserver and interobserver reproducibility of RAL measurement using Scheimpflug camera were evaluated with limit of agreement (LoA) and intraclass correlation coefficient (ICC). Consistency between RAL measurement of Scheimpflug camera and AS-OCT were assessed with LoA, correlation analysis and linear regression. RESULTS: For all subjects, intraobserver (LoA: -0.25 to 0.23 mm, ICC: 0.996) and interobserver reproducibility (LoA: -0.85 to 0.92 mm, ICC: 0.947) of RAL were good using Scheimpflug imaging. Both AS-OCT and Scheimpflug imaging found that the age-related cataract participants had smaller RAL (P=0.010, P=0.001 respectively). LoA of RAL measurement between AS-OCT and Scheimpflug imaging was -3.83 to -0.79 mm, and the Pearson correlation efficient was 0.909 (P<0.001). The RAL values measured by AS-OCT were significantly greater than that by Scheimpflug camera with a mean difference of 2.31 mm for all participants (P<0.001). The RAL measurement could be converted using the equation: YCASIA 2 =1.155 × XPentacam + 1.060. CONCLUSIONS: Both Scheimpflug camera system with internal caliper tool and the AS-OCT are fast and non-contact tools that could measure RAL successfully. The two measurement results are highly correlated and interchangeable through linear regression equation.