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BACKGROUND: Protozoan parasites of the genus Eimeria are the causative agents of chicken coccidiosis. Parasite resistance to most anticoccidial drugs is one of the major challenges to controlling this disease. There is an urgent need for a molecular marker to monitor the emergence of resistance against anticoccidial drugs, such as decoquinate. METHODS: We developed decoquinate-resistant strains by successively exposing the Houghton (H) and Xinjiang (XJ) strains of E. tenella to incremental concentrations of this drug in chickens. Additionally, we isolated a decoquinate-resistant strain from the field. The resistance of these three strains was tested using the criteria of weight gain, relative oocyst production and reduction of lesion scores. Whole-genome sequencing was used to identify the non-synonymous mutations in coding genes that were highly associated with the decoquinate-resistant phenotype in the two laboratory-induced strains. Subsequently, we scrutinized the missense mutation in a field-resistant strain for verification. We also employed the AlphaFold and PyMOL systems to model the alterations in the binding affinity of the mutants toward the drug molecule. RESULTS: We obtained two decoquinate-resistant (DecR) strains, DecR_H and XJ, originating from the original H and XJ strains, respectively, as well as a decoquinate-resistant E. tenella strain from the field (DecR_SC). These three strains displayed resistance to 120 mg/kg decoquinate administered through feed. Through whole-genome sequencing analysis, we identified the cytochrome b gene (cyt b; ETH2_MIT00100) as the sole mutated gene shared between the DecR_H and XJ strains and also detected this gene in the DecR_SC strain. Distinct non-synonymous mutations, namely Gln131Lys in DecR_H, Phe263Leu in DecR_XJ, and Phe283Leu in DecR_SC were observed in the three resistant strains. Notably, these mutations were located in the extracellular segments of cyt b, in close proximity to the ubiquinol oxidation site Qo. Drug molecular docking studies revealed that cyt b harboring these mutants exhibited varying degrees of reduced binding ability to decoquinate. CONCLUSIONS: Our findings emphasize the critical role of cyt b mutations in the development of decoquinate resistance in E. tenella. The strong correlation observed between cyt b mutant alleles and resistance indicates their potential as valuable molecular markers for the rapid detection of decoquinate resistance.
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Coccidiose , Decoquinato , Eimeria tenella , Parasitos , Doenças das Aves Domésticas , Animais , Eimeria tenella/genética , Decoquinato/farmacologia , Citocromos b/genética , Galinhas/parasitologia , Mutação de Sentido Incorreto , Simulação de Acoplamento Molecular , Resistência a Medicamentos/genética , Coccidiose/veterinária , Coccidiose/parasitologia , Mutação , Doenças das Aves Domésticas/parasitologiaRESUMO
OBJECTIVE: The objectives of this study were to analyze the difference between the preoperative radiological and postoperative pathological stages of colorectal cancer (CRC) and explore the feasibility of elastic lamina invasion (ELI) as a prognostic marker for patients with stage III colon cancer. METHODS: A total of 105 consecutive patients underwent radical surgery (R0 resection) for stage III colon cancer at the Cancer Hospital of China Medical University between January 2015 and December 2017. Clinicopathological features, including radiological stage and elastic lamina staining, were analyzed for prognostic significance in stage III colon cancer. RESULTS: A total of 105 patients with stage III colon cancer who met the criteria and had complete data available were included. The median follow-up period of survivors was 41 months. During the follow-up period, 33 (31.4%) patients experienced recurrence after radical resection, and the 3-year disease-free survival (DFS) rate was 64.8%. The consistency between preoperative radiological and postoperative pathological staging was poor (κ = 0.232, P < 0.001). The accuracy of ≤ T2 stage diagnoses was 97.1% (102/105), that of T3 stage was 60.9% (64/105), that of T4a stage was 68.6% (72/105) and that of T4b stage was 91.4% (96/105). The DFS rate of T3 ELI (+) patients was significantly lower than that of both T3 ELI (-) patients (P = 0.000) and pT4a patients (P = 0.013). The DFS rate of T3 ELI (-) patients was significantly higher than that of pT4b patients (P=0.018). T3 ELI (+) (HR (Hazard ratio), 8.444 [95% CI, 1.736-41.067]; P = 0.008), T4b (HR, 57.727[95% CI, 5.547-600.754]; P = 0.001), N2 stage (HR, 10.629 [95% CI, 3.858-29.286]; P < 0.001), stage III (HR, 0.136 [95% CI, 0.31-0.589]; P = 0.008) and perineural invasion (PNI) (HR, 8.393 [95% CI, 2.094-33.637]; P = 0.003) were independent risk factors for postoperative recurrence of stage III colon cancer. CONCLUSIONS: The consistency between preoperative radiological and postoperative pathological staging was poor, especially for tumors located in the ascending colon and descending colon. Elastic lamina staining is expected to become a stratified indicator of recurrence risk for patients with stage III colon cancer and a guide for individualized adjuvant chemotherapy, thus improving patient prognosis.
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Neoplasias do Colo , Humanos , Prognóstico , Estadiamento de Neoplasias , Invasividade Neoplásica/patologia , Estudos Retrospectivos , Neoplasias do Colo/patologiaRESUMO
The potential of Eimeria parasites as live vaccine vectors has been reported with successful genetic manipulation on several species like E. tenella, E. mitis and E. necatrix. Among seven Eimeria species infecting chickens, E. acervulina is a highly prevalent, moderately pathogenic species. Thus, it is valuable for the study of transfection and for use as a potential as vaccine vector. In this study, a plasmid containing expression cassette with enhanced yellow fluorescent protein (EYFP), red fluorescent protein (RFP) and 12 copies of extracellular domain of H9N2 avian influenza virus M2 (M2e) protein was used for the transfection. Nucleofected sporozoites were inoculated into birds through wing vein. Recombinant E. acervulina oocysts with 0.1% EYFP+ and RFP+ populations were collected from the feces of the inoculated birds. The fluorescent rate of transgenic parasites reached over 95% after nine successive propagations with a pyrimethamine selection in vivo and fluorescent-activated cell sorting (FACS) of progeny oocysts. The expression of M2e in the transgenic parasites (EaM2e) was confirmed by Western blot and its cytoplasm localization in sporozoites was displayed by an indirect immunofluorescent assay (IFA). Meanwhile, we found that the fecundity of EaM2e was equivalent to that of wild type E. acervulina (EaWT). Taken together, the stable transfection of E. acervulina was successfully established. Future studies will focus on whether transgenic E. acervulina can serve as a live vaccine vector.
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The fibrinogen-to-albumin ratio (FAR), reflecting the systemic coagulation, nutritional and inflammation status of patients, has matured into a prognostic marker for several tumor types. However, only a few studies have assessed the utility of the FAR as a prognostic indicator in patients with advanced gastric cancer (GC) receiving first-line chemotherapy. In the present study, 273 patients with advanced GC who received first-line chemotherapy between January 2014 and January 2019 at the Cancer Hospital of China Medical University (Shenyang, China) were retrospectively analyzed. Using the cut-off values determined by receiver operating characteristic (ROC) analysis, the patients were divided into low-FAR (≤10.03) and high-FAR (>10.03), low-fibrinogen (<3.8 g/l) and high-fibrinogen (≥3.8 g/l), and low-albumin (<40.55 g/l) and high-albumin (≥40.55 g/l) groups. The associations of the pretreatment FAR and clinicopathological characteristics with progression-free survival (PFS) and overall survival (OS) were evaluated. In order to estimate the prognostic value of the FAR for patients with poor prognosis or normal fibrinogen and albumin levels, subgroup analyses were performed. The FAR had a higher area under the ROC curve (0.690; 95% CI: 0.628-0.752; P<0.001) compared with either fibrinogen or albumin alone, which are common indicators of coagulation, nutritional and inflammatory indices. A high FAR was significantly associated with a more advanced stage, peritoneal metastasis, increased CA72-4 levels and anemia (all P<0.05). On survival analysis, a low FAR was associated with a longer PFS and OS compared with a high FAR (202 vs. 130 days and 376 vs. 270 days, respectively; both P<0.001), while the hazard ratio (HR) and P-values of the FAR were lower compared with those of fibrinogen and albumin alone on multivariate analysis (PFS: HR=0.638, 95% CI: 0.436-0.932, P=0.020; OS: HR=0.568, 95% CI: 0.394-0.819, P=0.002). Subgroup analysis indicated that among patients with poor prognosis, including multiple metastases, TNM stage IV and abnormal CA72-4 levels, the FAR may be used as an accurate prognostic marker (all P<0.05), and may also reliably identify patients with poor prognosis among those with normal fibrinogen and albumin levels (all P<0.001). The FAR was indicated to be a valuable marker for predicting PFS and OS in patients with advanced GC receiving first-line chemotherapy and is superior to either fibrinogen or albumin alone.
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The supported catalyst featuring highly dispersed active phase on support is the most important kind of industrial catalyst. Extensive research has demonstrated the critical role (in catalysis) of the interfacial interaction/perimeter sites between the active phase and support. However, the supported catalyst prepared by traditional methods generally presents low interface density because of limit contact area. Here, an ion-exchange inverse loading (IEIL) method has been developed, in which the precursor of support is controllably deposited onto the precursor of active phase by ion-exchange reaction, leading to an active core surrounded (by support) catalyst with various structures. The unique surrounded structure presents not only high interface density and mutually changed interface but also high stability due to the physical isolation of active phase, revealing superior catalytic performances to the traditional supported catalysts, suggesting the great potential of this new surrounded catalyst as the upgrade of supported catalyst in heterogeneous catalysis.
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Tumor biopsy is the standard method for cancer diagnosis and provides an important sample for pathological assessment. With the development of precision medicine, liquid biopsies are now an important tool to detect molecular changes and tumor heterogeneity. In recent years, research related to circulating tumor DNA (ctDNA) has intensified due to its non-invasive, convenient, comprehensive, and safety characteristics. Herein, we provide a review describing the clinical applications and prospects of ctDNA in colorectal cancer (CRC) diagnosis, monitoring and prognosis.
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Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.
Assuntos
Núcleo Celular/metabolismo , Galinhas/parasitologia , Eimeria/fisiologia , Parasitos/fisiologia , Transfecção , Animais , Eimeria/citologia , Injeções Intravenosas , Merozoítos/citologia , Merozoítos/fisiologia , Doenças das Aves Domésticas/parasitologia , Esporozoítos/fisiologiaRESUMO
Coccidiosis is a serious threat to the poultry industry, resulting in substantial economic losses worldwide. The effective development of alternative treatments for coccidiosis that does not involve chemotherapy drugs and does not result in antibiotic resistance relies on gaining a clearer understanding of the interaction between host intestinal microbiota and enteric coccidia. Here, we established an Eimeria tenella infection model in chickens and subsequently monitored the changes in the overall intestinal microbiome using 16S rRNA gene sequencing. We found that the gut (i.e. fecal) microbiota of infected chicken differed from that of uninfected naïve animals. Levels of non-pathogenic bacteria, including Lactobacillus and Faecalibacterium declined, whereas those of pathogenic bacteria, including Clostridium, Lysinibacillus, and Escherichia, increased over time in response to E. tenella infection. Similar dynamic changes of the fecal microbiota were observed in both Arbor Acres broilers and White Leghorn chickens, indicating that the perturbation of the microbiota was directly induced by E. tenella infection. Our findings could be used to further elucidate the serious damage to host health caused by coccidia infection, leading to the development of new effective treatment options for coccidiosis.
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Galinhas/parasitologia , Eimeria tenella/patogenicidade , Microbioma Gastrointestinal/genética , Oocistos/fisiologia , Animais , Ceco/parasitologia , Ceco/patologia , Coccidiose/parasitologia , Eimeria tenella/isolamento & purificação , Fezes/parasitologia , Doenças das Aves Domésticas/parasitologia , Doenças das Aves Domésticas/patologia , RNA Ribossômico 16S/genéticaRESUMO
Proton-exchange membrane fuel cells are one kind of renewable and clean energy conversion device, whose metallic bipolar plates are one of the key components. However, high interfacial contact resistance and poor corrosion resistance are still great challenges for the commercialization of metallic bipolar plates. In this study, we demonstrated a novel strategy for depositing TiC x/amorphous carbon (a-C) nanolayered coatings by synergy of 60 and 300 V bias voltage to enhance corrosion resistance and interfacial conductivity. The synergistic effects of bias voltage on the composition, microstructure, surface roughness, electrochemical corrosion behaviors, and interfacial conductivity of TiC x/a-C coatings were explored. The results revealed that the columnar structures in the inner layer were suppressed and the surface became rougher with the 300 V a-C layer outside. The composition analysis indicated that the sp2 content increased with an increase of 300 V sputtering time. Due to the synergy strategy of bias voltage, lower corrosion current densities were achieved both in potentiostatic polarization (1.6 V vs standard hydrogen electrode) and potentiodynamic polarization. With the increase of 300 V sputtering time, the interfacial conductivity was improved. The enhanced corrosion resistance and interfacial conductivity of the TiC x/a-C coatings would provide new opportunities for commercial bipolar plates.