No abstract

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.

Melon (Cucumis melo) is an annual herbaceous plant of the family Cucurbitaceae. Phytophthora rot, caused by Phytophthora capsici is a serious threat to cucurbits crops production as it directly infects the host plant, and it is difficult to control because of variable pathogenicity. This study investigated the resistance of 450 accessions of melon germplasm against Phytophthora rot by inoculating the seedlings with sporangial suspension (10 5 or 6 zoosporangia/ml) of P. capsici. Disease incidence of Phytophthora rot was observed on the melon germplasm at 7-day intervals for 35 days after inoculation. Susceptible melon germplasm showed either severe symptoms of stem and root rot or death of the whole plant. Twenty out of 450 tested accessions showed less than 20% disease incidence, of which five accessions showed a high level of resistance against Phytopthtora rot. Five resistant accessions, namely IT119813, IT138016, IT174911, IT174927, and IT906998, scored 0% disease incidence under high inoculum density of P. capsici (10 6 zoosporangia/ mL). We recommend that these candidate melon germplasm may be used as genetic resources in the breeding of melon varieties resistant to Phytophthora rot.

Objectives To investigate and validate potential standard compounds for standardization of Sinbaro3 pharmacopuncture prepared at OO Hospital of Korean Medicine. Methods Sinbaro3 pharmacopuncture was prepared by extraction, purification and hydrolysis of Harpagophytum procumbens, and various potential standard compounds were quantified through HPLC-UV and HPLC-MS analysis. Validation was examined by assessing specificity, linearity, precision, and accuracy. Results The retention time of harpagide and cinnamic acid were 15.2 min and 28.2 min, respectively, and both showed good linearity in analysis by concentration at 0.9999 and 0.9998, respectively. Intra-day variation of precision was 0.0015~0.0045% and 0.0058~ 0.1629%, while inter-day variation of precision was 0.0011~0.0243% and 0.0098~0.1629%, and that of accuracy was 99.53~99.89% and 99.50~99.91%, respectively. Conclusions Harpagide and cinnamic acid, which are hydrolyzates of harpagoside within Sinbaro3 pharmacopuncture, were both validated using HPLC-MS and HPLC-UV analysis, and Sinbaro3 pharmacopuncture contained 78.41 ug/ml harpagide, and 2.05 ug/ml cinnamic acid. .

Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.