Clinical trials employing AAV vectors for hemophilia A have been hindered by unanticipated immunological and/or inflammatory responses in some of the patients. Also, these trials have often yielded lower levels of transgene expression than were expected based upon preclinical studies, highlighting the poor correlation between the transduction efficiency observed in traditional 2D cultures of primary cells in vitro, and that observed in those same cell types in vivo. It has been also recognized that there are marked species-specific differences in AAV-vector tropism, raising the critical question of the accuracy with which various animal models will likely predict tropism/vector transduction efficiency, and eventual treatment success in humans. Human liver tissue equivalents (hLTEs) are comprised of major cell types in the liver in physiologically relevant frequencies and possess the ability to recapitulate the biology and function of native human liver. Here, we hypothesize that hLTEs can be used as a better model to predict the efficacy and safety of AAV gene therapy in humans. We fabricated hLTEs using 75% hepatocytes, 10% stellate cells, 10% Kupffer cells, and 5% liver sinusoid-derived endothelial cells in 96-well Elplasia plates with 79 microwells per well. hLTEs were transduced at an MOI of 10 5vg/cell, on the day of fabrication, with the clinically relevant serotypes AAV5 (hLTE-5) or AAV3b (hLTE-3b), both encoding a GFP reporter. After 4 days of self-aggregation, live/dead assay was performed to confirm viability. Non-transduced hLTEs served as negative controls (hLTE(-)), and hLTEs exposed to 20 mM acetaminophen were used as positive controls for liver inflammation/damage. Incucyte® Live-Cell Imaging system was used to track the aggregation and GFP expression of hLTEs. Over the course of the next 5 days, media was collected to determine hepatic functionality, RNA was isolated to assess dysregulation of genes involved in inflammation and fibrosis, DNA was isolated to determine whether AAV vectors integrate into the genome of human hepatocytes and, if so, to define the frequency at which this occurs and the genomic loci of integration, and hLTEs were fixed and processed at appropriate times for histological analyses and transmission electron microscopy (TEM). TEM analysis revealed that all groups exhibited microvilli and bile-canaliculus-like structures, demonstrating the formation of a rudimentary biliary system and, more importantly, proving that hLTEs resemble native liver structure. Incucyte® imaging showed that AAV5 and AAV3b transduction impaired formation of hLTEs (57.57 ± 2.42 and 24.57 ± 4.01 spheroids/well, respectively) in comparison with hLTE(-) (74.86 ± 3.8 spheroids/well). Quantification of GFP expression demonstrated that AAV5 yielded the most efficient transduction of hLTEs (fold change in GFP expression compared to control: 2.73 ± 0.09 and 1.19 ± 0.03 for hLTE-5 and hLTE-3b, respectively). Chromogenic assays showed decreased urea production in cell culture supernatants of AAV transduced groups compared to the non-transduced hLTEs on days 6 and 10 of culture, demonstrating decreased hepatocyte functionality. However, ALT and AST levels were similar in all groups. On day 10, hLTEs were either used for RNA isolation or fixed in 4% PFA and processed for histology. Masson's Trichrome and Alcian Blue/Sirius Red staining was performed to detect fibrosis, which was then quantified using ImageJ. These analyses showed no significant increase in fibrosis in either hLTE-5 or hLTE-3b compared to hLTE(-). Nevertheless, RT 2 PCR Array for Human Fibrosis detected dysregulation of several genes involved in fibrosis/inflammation in both hLTE-5 and hLTE-3b (16/84 and 26/84, respectively). In conclusion, data collected thus far show successful recapitulation of native liver biology and demonstrate that AAV5 transduces hLTEs more efficiently than AAV3b. However, impaired self-aggregation and decreased hepatocyte functionality was observed in both AAV-transduced groups. Studies to address the incidence and location(s) of AAV integration are ongoing. We have thus shown that the hLTE system can provide critical new knowledge regarding the efficacy and safety of AAV gene therapy in the human liver. Disclosures No relevant conflicts of interest to declare.
Abstract:The Janzen–Connell hypothesis suggests that highly specific pathogens decrease seedling survival close to the parent plant; however, the underlying mechanism remains unclear. Here, we tested the host specificity of soil pathogens to germinating seeds of Cyclobalanopsis fleuryi and Cryptocarya chinensis in tropical montane rain forest and cloud forest on Hainan, south China. Rhizospheric soils surrounding eight adult trees per species were collected in each forest type and divided into five soil treatments: parent, fungicide-sterilized, autoclave-sterilized, Fusarium-added and Pythium-added soils. Surface-sterilized seeds were sown in each of the five soil treatments and grown in two forest types. The seed germination percentages were significantly higher in sterilized soils (C. fleuryi, 41.5%; C. chinensis, 29.4%) than in non-sterilized soils (C. fleuryi, 28.3%; C. chinensis, 17.1%) in montane rain forest. The seed germination percentages in rhizospheric soil of conspecific parent trees were significantly lower in montane rain forest (C. fleuryi, 17.3%; C. chinensis, 10.5%) than in cloud forest (C. fleuryi, 37.1%; C. chinensis, 21.1%). Our results also suggest that the level of pathogen activity in each tree species varies depending on the environment. Our results support the hypothesis that host-specific pathogens shape tree species composition by differentially affecting seed germination under different environmental conditions.
The failure behavior and the vulnerable position of the rigid frame bridge are analyzed by nonlinear response analysis, based on incremental dynamic analysis (IDA). The damage index, applying for long-span continuous rigid frame bridge with high pier, is studied. The seismic vulnerability problem of the whole bridge is analyzed by fiber model. The strain was used for damage index of piers. The displacement was used for damage index of bearings. According to the studies above, the fragility curves are established based on integral performance of the bridge.
An experimental investigation was conducted to analyze the machined surface in high-speed dry milling of Ti-6Al-4V alloy using coated carbide inserts, with white light interferometer, scanning electron microscope (SEM), and X-ray diffraction (XRD) employed. The effect of cutting force and the workpiece temperature rise on the machined surface under different cutting speeds was discussed. As cutting speed increases above 150 m/min, the mean cutting forces decrease remarkably, but the corresponding higher temperature will be harmful to the machined surface. A deformed layer is detected by SEM with grains orientation along the feed direction from the sub-surface microstructure. The 3D surface topography and XRD patterns confirm the intense deformation of the machined surface and show a crystallographic texture modification. However, no phase transformation was observed. The β phases seem to experience more deformation and volume shrinkage in the near surface with the increase in cutting speed. And the observed variations of the machined surface with the cutting speed should be attributed to the elevated workpiece temperature rise under dry milling.
In terms of good optical properties and high thermal stability, Mo/Si3N4/Mo/Si3N4/SiO2 coatings based on metal/dielectric multilayer structure were adapted to the solar selective coating at high operating temperatures. The coatings exhibited high solar absorptance in the range of 0.924 ~ 0.936 and low thermal emittance of 0.114 ~ 0.118. The coatings deposited on quartz substrates were thermally stable up to 625 °C in air for 2 h, while they were degraded at 650 °C from the characterization of the absorptance and emittance. The degradation of the coatings was mainly due to the oxidation of molybdenum in air, which was confirmed by Raman spectroscopy. Compared with the thermal stability in air, the coatings were much more stable in vacuum under high temperature. The remarkable thermal stability of the Mo/Si3N4/Mo/Si3N4/SiO2 coatings in air and in vacuum makes them suitable to be applied at high temperature applications.
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