METHODS: We used transmission electron microscopy (TEM) to investigate post-mortem tissue sections of patients with clinical melioidosis to identify the localisation of a recently identified gut microbiome, B. pseudomallei within host cells. The intranuclear presence of B. pseudomallei was confirmed using transmission electron microscopy (TEM) of experimentally infected guinea pig spleen tissues and Live Z-stack, and ImageJ analysis of fluorescence microscopy analysis of in vitro infection of A549 human lung epithelial cells.
RESULTS: TEM investigations revealed intranuclear localization of B. pseudomallei in cells of infected human lung and guinea pig spleen tissues. We also found that B. pseudomallei induced actin polymerization following infection of A549 human lung epithelial cells. Infected A549 lung epithelial cells using 3D-Laser scanning confocal microscopy (LSCM) and immunofluorescence microscopy confirmed the intranuclear localization of B. pseudomallei.
CONCLUSION: B. pseudomallei was found within the nuclear compartment of host cells. The nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrrent episodes or recrudescence of infection.
METHODS: We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca2+-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes.
RESULTS: Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca2+-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca2+ elevations and a disorganized cytoskeleton.
CONCLUSIONS: The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca2+ may contribute to structural damage in the podocytes.
MATERIALS AND METHOD: Eighty-seven paraffin-embedded ameloblastoma cases (20 unicystic, 47 solid/multicystic, 3 desmoplastic and 17 recurrent) were subjected to immunohistochemistry for expression of cortactin, N-WASP, WIP, Src kinase and F-actin, and findings correlated with clinicopathological parameters.
RESULTS: Invadopodia proteins (except Src kinase) and F-actin were widely detected in ameloblastoma (cortactin: n = 73/87, 83.9%; N-WASP: n = 59/87; 67.8%; WIP: n = 77/87; 88.5%; and F-actin: n = 87/87, 100%). Protein localization was mainly cytoplasmic and/or membranous, and occasionally nuclear for F-actin. Cortactin, which functions as an actin-scaffolding protein, demonstrated significantly higher expression levels within ameloblastoma tumoral epithelium than in stroma (P < 0.05). N-WASP, which coordinates actin polymerization and invadopodia-mediated extracellular matrix degradation, was overexpressed in the solid/multicystic subtype (P < 0.05). WIP, an upstream regulator of N-WASP, and F-actin were significantly upregulated along the tumour invasive front compared to tumour centres (P < 0.05). Except for males with cortactin overexpression, other clinical parameters (age, ethnicity and anatomical site) showed no significant correlations.
CONCLUSIONS: Present results suggest that local invasiveness of ameloblastoma is dependent upon the migratory potential of its tumour cells as defined by their distribution of cortactin, N-WASP and WIP in correlation with F-actin cytoskeletal dynamics.
Materials and Methods: All groups of hFOB 1.19 cells were induced with 12.5 μg/ml of BPA except the control (Ctrl) group. Meanwhile, treated groups received phytoestrogens; Daidzein (Dz), Genistein (Gt), Equol (Eq) and 17β-oestradiol (Est) in different concentrations for 24 hr duration.
Results: We found that the protein expression of non-classical oestrogen-related receptor (ERRG) was highly expressed in BPA group, whereas classical oestrogen receptor alpha (ERα) and oestrogen receptor beta (ERβ) were relatively increased with phytoestrogens treatment under BPA exposure. The dense actin cytoskeletal filaments were also observed. qRT-PCR showed up-regulation of mitogen-activated protein kinase 3 (MAPK3) and G protein-coupled receptor 30 (GPR30) expressions; significant down-regulation of ERRG and up-regulation of ERα and ERβ were observed in phytoestrogens-treated cells, which was supported by the increased expressions of oestrogen receptor 1 (ESR1) and oestrogen receptor 2 (ESR2).
Conclusion: Phytoestrogens improved the deteriorative effect of BPA via down-regulation of ERRG in hFOB 1.19 cells. This study showed that the efficacy of consumption of phytoestrogens in rendering them as potential therapeutic strategy in combating the adverse bone effects of BPA.
METHODS: This study was conducted between the years 2014 to 2016 at the Tissue Engineering Centre, UKM Medical Centre. OTC-I was extracted from ovine tendon, and fabricated into 3D scaffolds in the form of sponge, hydrogel and film. A polystyrene surface coated with OTC-I was used as the 2D culture condition. Genipin was used to crosslink the OTC-I. A non-coated polystyrene surface was used as a control. The mechanical strength of OTC-I scaffolds was evaluated. Attachment, proliferation and morphological features of HDF were assessed and compared between conditions.
RESULTS: The mechanical strength of OTC-I sponge was significantly higher than that of the other scaffolds. OTC-I scaffolds and the coated surface significantly enhanced HDF attachment and proliferation compared to the control, but no differences were observed between the scaffolds and coated surface. In contrast, the morphological features of HDF including spreading, filopodia, lamellipodia and actin cytoskeletal formation differed between conditions.
CONCLUSION: OTC-I can be moulded into various scaffolds that are biocompatible and thus could be suitable as scaffolds for developing tissue substitutes for clinical applications and in vitro tissue models. However, further study is required to determine the effect of morphological properties on the functional and molecular properties of HDF.