A rhoptry protein, localizing in the bulb region of rhoptries, could induce protective immunity against Eimeria tenella infection.

Background: Rhoptry organelle proteins (ROPs) secreted by apicomplexan parasites play important roles during parasites invasion and survival in host cells, and are potential vaccine candidates against apicomplexan diseases. Eimeria tenella (E. tenella) is one of the most noteworthy apicomplexan species, which causes hemorrhagic pathologies. Although dozens of putative E. tenella ROP sequences are annotated, most ROP proteins are not well studied. Methods: In this study, an E. tenella ROP21 gene was identified and the recombinant EtROP21 protein (rEtROP21) was expressed in Escherichia coli. The developmental expression levels, localization, and protective efficacy against E. tenella infection in chickens were studied. Results: An EtROP21 gene fragment with an open reading frame (ORF) of 981 bp was obtained from the Beijing strain of E. tenella. The rEtROP21 has a molecular weight of approximately 50 kDa and was recognized by rEtROP21-immunized mouse serum. Two specific protein bands, about 43 KDa and 95 KDa in size, were detected in the whole sporozoite proteins using the rEtROP21-immunized chicken serum. RT-qPCR analysis of the E. tenella ROP21 gene (EtROP21) revealed that its mRNA levels were higher in merozoites and sporozoites than in sporulated and unsporulated oocysts. Immunofluorescence and immunoelectron analyses showed that the EtROP21 protein predominantly localizes in the bulb region of rhoptries distributed at anterior, posterior, and perinuclear regions of E. tenella sporozoites. Immunization and challenge experiments revealed that immunizing chickens with rEtROP21 significantly increased their average body weight gain while decreasing mean lesion score and oocyst output (P <0.05). When compared with the challenged control group, the rEtROP21-immunized group was associated with a significantly higher relative weight gain (90.2%) and a greater reduction in oocyst output (67%) (P <0.05). The anticoccidial index of the rEtROP21-immunized group was 163.2. Chicken serum ELISA revealed that the levels of the specific anti- rEtROP21 antibody, IFN-γ, and IL-4 were significantly higher in the rEtROP21-immunized group than in the challenged control group (P <0.05). Conclusion: These results indicate that rEtROP21 can induce a high level of specific immune response and it is a potential candidate for the development of vaccines against E. tenella infection in chickens.

Multifunctional O-phenanthroline silver(I) complexes for antitumor activity against colorectal adenocarcinoma cells and antimicrobial properties by multiple mechanisms.

A series of O-phenanthroline silver(I) complexes were synthesized and characterized by infrared (IR) spectroscopy, mass spectrometry (MS), 1H nuclear magnetic resonance (NMR) spectroscopy and single-crystal X-ray crystallography. The cytotoxicity of the silver(I) complex (P-131) was evaluated in the cancer cell lines HCT-116, HeLa, and MDA-MB-231 and the normal cell line LO2 via MTT assays. The 50% inhibition concentration (IC50) of P-131 on HCT116 cell line is 0.86 ± 0.03 µM. It is far lower than the IC50 value of cisplatin (9.08 ± 1.10 µM), the IC50 value of normal cell LO2 (76.20 ± 0.48 µM) is much higher than that of cisplatin (3.99 ± 0.74 µM), indicating that its anticancer effect is stronger than that of cisplatin, and its biological safety is greater than that of cisplatin. Furthermore, anticancer mechanistic studies showed that P-131 inhibited cell proliferation by blocking DNA synthesis and acted temporally on the nucleus in dividing HCT-116 cells. Moreover, P-131 increased intracellular reactive oxygen species (ROS) levels in a dose-dependent manner. Notably, 10 mg/kg P-131 showed better antitumor effects than oxaliplatin in an HCT116 human colorectal xenograft mouse model without inducing toxicity. Moreover, the microdilution broth method was used to evaluate the antimicrobial properties of P-131 against Pseudomonas aeruginosa and Candida albicans. A biofilm eradication study was also performed using the crystal violet method and confocal laser scanning microscopy.

(8-Hydroxyquinoline) Gallium(III) Complex with High Antineoplastic Efficacy for Treating Colon Cancer via Multiple Mechanisms.

A series of (8-hydroxyquinoline) gallium(III) complexes (CP-1-4) was synthesized and characterized by single X-ray crystallography and density functional theory (DFT) calculation. The cytotoxicity of the four gallium complexes toward a human nonsmall cell lung cancer cell line (A549), human colon cancer cell line (HCT116), and human normal hepatocyte cell line (LO2) was evaluated using MTT assays. CP-4 exhibited excellent cytotoxicity against HCT116 cancer cells (IC50 = 1.2 ± 0.3 µM) and lower toxicity than cisplatin and oxaliplatin. We also evaluated the anticancer mechanism studies in cell uptake, reactive oxygen species analysis, cell cycle, wound-healing, and Western blotting assays. The results showed that CP-4 affected the expression of DNA-related proteins, which led to the apoptosis of cancer cells. Moreover, molecular docking tests of CP-4 were performed to predict other binding sites and to confirm its higher binding force to disulfide isomerase (PDI) proteins. The emissive properties of CP-4 suggest that this complex can be used for colon cancer diagnosis and treatment, as well as in vivo imaging. These results also provide a foundation for the development of gallium complexes as potent anticancer agents.

Gallium Metal-Organic Nanoparticles with Albumin-Stabilized and Loaded Graphene for Enhanced Delivery to HCT116 Cells.

Background: Gallium (III) metal-organic complexes have been shown to have the ability to inhibit tumor growth, but the poor water solubility of many of the complexes precludes further application. The use of materials with high biocompatibility as drug delivery carriers for metal-organic complexes to enhance the bioavailability of the drug is a feasible approach. Methods: Here, we modified the ligands of gallium 8-hydroxyquinolinate complex with good clinical anticancer activity by replacing the 8-hydroxyquinoline ligands with 5-bromo-8-hydroxyquinoline (HBrQ), and the resulting Ga(III) + HBrQ complex had poor water solubility. Two biocompatible materials, bovine serum albumin (BSA) and graphene oxide (GO), were used to synthesize the corresponding Ga(III) + HBrQ complex nanoparticles (NPs) BSA/Ga/HBrQ NPs and GO/Ga/HBrQ NPs in different ways to enhance the drug delivery of the metal complex. Results: Both of BSA/Ga/HBrQ NPs and GO/Ga/HBrQ NPs can maintain stable existence in different solution states. In vitro cytotoxicity test showed that two nanomedicines had excellent anti-proliferation effect on HCT116 cells, which shown higher level of intracellular ROS and apoptosis ratio than that of cisplatin and oxaliplatin. In addition, the superior emissive properties of BSA/Ga/HBrQ NPs and GO/Ga/HBrQ NPs allow their use for in vivo imaging showing highly effective therapy in HCT116 tumor-bearing mouse models. Conclusion: The use of biocompatible materials for the preparation of NPs against poorly biocompatible metal-organic complexes to construct drug delivery systems is a promising strategy that can further improve drug delivery and therapeutic efficacy.

Evaluation of porcine circovirus type 2 infection in in vitro embryo production using naturally infected oocytes.

In vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) are important breeding techniques for livestock. High-quality MII oocytes produced from in vitro maturation (IVM) are required for the two techniques listed above. The ovaries used for IVM operations are primarily acquired from commercial abattoirs, and the pathogen status of slaughtered animals becomes an unavoidable issue. Our previous monitoring data showed that porcine circovirus type 2 (PCV-2) is the main pathogen present in ovaries from abattoirs. However, the characteristics and effects of PCV-2 infection in oocyte maturation and in vitro production (IVP) of embryos are unclear, and currently there are no relevant studies. Therefore, the aim of this study was to determine the PCV-2 infection pattern and determine whether it affects oocyte in vitro maturation and IVP embryo development. More than five hundred ovaries and five thousand oocytes were utilized in the present study. Polymerase chain reaction (PCR) was used to detect PCV-2 DNA in ovaries, follicular fluid (FF), oocytes, cumulus cells and IVP embryos. The effects of viral infections on the rate of oocyte maturation and IVP embryo development were evaluated. We also analyzed the number of copies of the virus in the IVM and IVP process by absolute quantitative fluorescence PCR. Our study showed that the prevalent virus subgenotype in ovaries was PCV-2a. PCV-2a infection did not significantly affect ovarian/oocyte morphology and maturation. Moreover, virus infection did not have a significant effect on the development of the IVP embryos except for a reduction in IVF blastocyst cell numbers. Further tests showed that the viral copy numbers fluctuated at different stages between the IVP embryos and culture medium. For the first time, this study identified the infection pattern of naturally sourced PCV-2 in the course of oocyte maturation and embryo development.