Methods: In this study, comparative genome analysis was carried out using the G. boninense NJ3 genome to identify and characterize carbohydrate-active enzyme (CAZymes) including CWDE in the fungal genome. Augustus pipeline was employed for gene identification in G. boninense NJ3 and the produced protein sequences were analyzed via dbCAN pipeline and PhiBase 4.5 database annotation for CAZymes and plant-host interaction (PHI) gene analysis, respectively. Comparison of CAZymes from G. boninense NJ3 was made against G. lucidum, a well-studied model Ganoderma sp. and five selected pathogenic fungi for CAZymes characterization. Functional annotation of PHI genes was carried out using Web Gene Ontology Annotation Plot (WEGO) and was used for selecting candidate PHI genes related to cell wall degradation of G. boninense NJ3.
Results: G. boninense was enriched with CAZymes and CWDEs in a similar fashion to G. lucidum that corroborate with the lignocellulolytic abilities of both closely-related fungal strains. The role of polysaccharide and cell wall degrading enzymes in the hemibiotrophic mode of infection of G. boninense was investigated by analyzing the fungal CAZymes with necrotrophic Armillaria solidipes, A. mellea, biotrophic Ustilago maydis, Melampsora larici-populina and hemibiotrophic Moniliophthora perniciosa. Profiles of the selected pathogenic fungi demonstrated that necrotizing pathogens including G. boninense NJ3 exhibited an extensive set of CAZymes as compared to the more CAZymes-limited biotrophic pathogens. Following PHI analysis, several candidate genes including polygalacturonase, endo β-1,3-xylanase, β-glucanase and laccase were identified as potential CWDEs that contribute to the plant host interaction and pathogenesis.
Discussion: This study employed bioinformatics tools for providing a greater understanding of the biological mechanisms underlying the production of CAZymes in G. boninense NJ3. Identification and profiling of the fungal polysaccharide- and lignocellulosic-degrading enzymes would further facilitate in elucidating the infection mechanisms through the production of CWDEs by G. boninense. Identification of CAZymes and CWDE-related PHI genes in G. boninense would serve as the basis for functional studies of genes associated with the fungal virulence and pathogenicity using systems biology and genetic engineering approaches.
DESIGN: We employed enzymatic digestion of cartilage using collagenase II and trypsin. The chondrocytes yield, growth kinetics, aggrecan, and collagen type 2 (COL2) expression were evaluated. Collagen type 1 (COL1) mRNA expression was assessed to monitor the possibility of chondrocytes dedifferentiation.
RESULTS: Chondrocyte yield per gram of cartilage was significantly higher (P < 0.05) using collagenase II in Hank's balanced salt solution (HBSS) compared with 0.25% trypsin. The number of chondrocyte yield per gram was higher in cartilage digested with collagenase in HBSS compared with Dulbecco's modified Eagle medium/F12; however, the difference was not statistically significant. Chondrocytes seeded at lower densities had shorter population doubling time compared to those seeded at higher density. Protein and gene expression of chondrocyte phenotype indicates the expression of aggrecan and COL2. The expression of COL1 was significantly increased (P < 0.05) in passage 3 compared with primary chondrocytes. The mRNA expression of chondrocyte phenotype was similar in primary and passaged one cells.
CONCLUSIONS: Collagenase in HBSS yield the highest number of viable chondrocytes and the isolated cells expressed chondrocyte phenotype. This protocol can be employed to generate large number of viable chondrocytes, particularly with limited cartilage biopsies.
OBJECTIVE: This study aims to evaluate the performance of Putralytica and Qure.ai software for CXR screening and PTB diagnosis among the Malaysian population.
METHODS: We will conduct a retrospective case-control study at the Respiratory Medicine Institute, National Cancer Institute, and Sungai Buloh Health Clinic. A total of 1500 CXR images of patients who completed treatments or check-ups will be selected and categorized into three groups: (1) abnormal PTB cases, (2) abnormal non-PTB cases, and (3) normal cases. These CXR images, along with their clinical findings, will be the reference standard in this study. All patient data, including sociodemographic characteristics and clinical history, will be collected prior to screening via Putralytica and Qure.ai software and readers' interpretation, which are the index tests for this study. Interpretation from all 3 index tests will be compared with the reference standard, and significant statistical analysis will be computed.
RESULTS: Data collection is expected to commence in August 2023. It is anticipated that 1 year will be needed to conduct the study.
CONCLUSIONS: This study will measure the accuracy of Putralytica and Qure.ai software and whether their findings will concur with readers' interpretation and the reference standard, thus providing evidence toward the effectiveness of implementing AI in the medical setting.
INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/36121.