Construction of a three-dimensional urothelium on-chip with barrier function based on urinary flow microenvironment.

The urothelium covers the inner surface of the urinary tract, forming a urinary tract barrier. Impairment of the integrity and dysfunction of the urinary tract barrier is associated with the occurrence and development of various diseases. The development of a three-dimensional model of the urothelium is critical for pathophysiological studies of this site, especially under physiological fluid shear stress stimulated by the urinary flow. In this study, a urothelium on-chip is fabricated with micromilling and replica molding techniques, which contains a microfluidic chip for cell culture and a pump-based fluid perfusion system. The mechanical properties of the human urinary tract are simulated by adjusting the concentration and degree of amino substitution of the gelatin methacrylate hydrogel. The matrix stiffness is similar to the natural urinary tract. Pulsatile flow and periodic flow are provided to simulate the fluid environment of the upper and lower urinary tracts, respectively. The results show that the physiological fluid shear stress could promote the differentiation and maturation of urothelial cells. The model could simulate the three-dimensional structure of urothelium and urinary flow microenvironment, showing morphological structure close to the natural urothelium, specific differentiation and maturation markers (uroplakin 2, cytokeratin 20), and urothelial barrier function.

Application of microfluidic chips in the simulation of the urinary system microenvironment.

The urinary system, comprising the kidneys, ureters, bladder, and urethra, has a unique mechanical and fluid microenvironment, which is essential to the urinary system growth and development. Microfluidic models, based on micromachining and tissue engineering technology, can integrate pathophysiological characteristics, maintain cell-cell and cell-extracellular matrix interactions, and accurately simulate the vital characteristics of human tissue microenvironments. Additionally, these models facilitate improved visualization and integration and meet the requirements of the laminar flow environment of the urinary system. However, several challenges continue to impede the development of a tissue microenvironment with controllable conditions closely resemble physiological conditions. In this review, we describe the biochemical and physical microenvironment of the urinary system and explore the feasibility of microfluidic technology in simulating the urinary microenvironment and pathophysiological characteristics in vitro. Moreover, we summarize the current research progress on adapting microfluidic chips for constructing the urinary microenvironment. Finally, we discuss the current challenges and suggest directions for future development and application of microfluidic technology in constructing the urinary microenvironment in vitro.

A high-throughput microplate toxicity screening platform based on Caenorhabditis elegans.

Caenorhabditis elegans (C. elegans), an established model organism, has been widely used in environmental toxicology research. However, most of the current toxicity testing methods based on worms are time-consuming. In this study we aimed to develop an automated and highly-integrated platform for high-throughput and in situ toxicity testing. Considering the superiority of C. elegans as a neurotoxicological model, this platform mainly evaluates general toxicology and neurotoxicology endpoints, which are usually induced by metals and pesticides, the major environmental contaminants. Microplates were used as a worm culturing system, which have good compatibility with any commercial microplate applicable instruments. We developed a microfluidic-based module for worm dispensing, and an image acquisition/analysis module for monitoring worms and detecting toxicity endpoints in bright filed. These were collectively incorporated with a commercial pipetting workstation for automated food/drug delivery and a high-content analysis system for fluorescence detection. The integrated platform achieved an efficient on-demand worm dispensing, long-term maintenance, regular monitoring and imaging, survival assay and behavioral analyses, and visualized gene reporter assay. Moreover, "Lab on Web" was achieved by connecting the platform to the web for remote operation, worm monitoring, and phenotype calculation. To demonstrate the ability of the platform for automated toxicity testing assays; worms were treated with cadmium and longevity, neurotoxicity, developmental toxicity and gst-4 expression were evaluated. We determined its feasibility and proposed the potential application in high-throughput toxicity screening for environmental risk assessment in the nearest future.

[Spatio-Temporal Variations and Source Apportionment of Carbonaceous Species in PM2.5 Across Multiple Sampling Locations in the Chengdu Plain].

To understand the characteristics and sources of carbonaceous aerosols, one-year PM2.5 samples were analyzed for their organic carbon (OC) and elemental carbon (EC) content, following the thermal/optical transmission protocol in three cities[Deyang (DY), Chengdu (CD), and Meishan (MS)] in the Chengdu Plain. The observed annual average concentrations (µg·m-3) were in the following order:MS (15.8±9.6 OC and 6.6±5.3 EC) > CD (13.0±7.5 OC and 4.7±3.6 EC) > DY (9.6±6.1 OC and 3.4±2.6 EC). Organic matter (1.6OC) and EC was regarded as the total carbonaceous aerosols (TCA) amount, and the TCA/PM2.5 ratios at the three above-mentioned cities were 36%, 34%, and 30% respectively. The EC-trace method was used to estimate secondary organic carbon (SOC), which accounted for 38%, 46%, and 47% of total OC in MS, CD, and DY. Daily variations of OC and EC concentrations exhibited significant daily variations, with simultaneous peaks on Oct. 12th to 13th, 2013, Dec. 2nd to 7th, 2013, and mid-to-late Jan., 2014. The surging concentrations of K+ during the pollution period implied the contribution of biomass burning to heavy pollution. Six sources were resolved by the positive matrix factorization (PMF) model, whose contributions to the total carbon (TC) were:biomass burning (46%-56%), secondary aerosols (26%-38%), vehicle emission (9%-12%), fugitive dust (3%-4%), coal combustion (2%-3%), and industry emission (1%-2%). Biomass burning activities presented a significant influence on TC throughout the year, especially in autumn and winter.

Leaf growth in early development is key to biomass heterosis in Arabidopsis.

Arabidopsis thaliana hybrids have similar properties to hybrid crops, with greater biomass relative to the parents. We asked whether the greater biomass was due to increased photosynthetic efficiency per unit leaf area or to overall increased leaf area and increased total photosynthate per plant. We found that photosynthetic parameters (electron transport rate, CO2 assimilation rate, chlorophyll content, and chloroplast number) were unchanged on a leaf unit area and unit fresh weight basis between parents and hybrids, indicating that heterosis is not a result of increased photosynthetic efficiency. To investigate the possibility of increased leaf area producing more photosynthate per plant, we studied C24×Landsberg erecta (Ler) hybrids in detail. These hybrids have earlier germination and leaf growth than the parents, leading to a larger leaf area at any point in development of the plant. The developing leaves of the hybrids are significantly larger than those of the parents, with consequent greater production of photosynthate and an increased contribution to heterosis. The set of leaves contributing to heterosis changes as the plant develops; the four most recently emerged leaves make the greatest contribution. As a leaf matures, its contribution to heterosis attenuates. While photosynthesis per unit leaf area is unchanged at any stage of development in the hybrid, leaf area is greater and the amount of photosynthate per plant is increased.

Cotyledons contribute to plant growth and hybrid vigor in Arabidopsis.

MAIN CONCLUSION: In hybrids of Arabidopsis, cotyledons influence the amount and proportion of hybrid vigor in total plant growth. We found Arabidopsis cotyledons are essential for plant growth and in some hybrids for hybrid vigor. In hybrids between C24 and Landsberg erecta (Ler), biomass vigor (heterosis) occurs in the first few days after sowing (DAS), with hybrid cotyledons being larger than those of their parents. C24xLer hybrids are ahead of their parents in activating photosynthesis and auxin pathway genes in cotyledons at 3-4 DAS. "Earliness" is also present in newly emerged C24xLer hybrid leaves. We showed cotyledon removal at 4 DAS caused significant biomass reduction in later growth in hybrids and parental lines. The biomass decrease caused by cotyledon removal can be partially rescued by exogenous sucrose or auxin with different genotypes responding to sucrose and/or auxin differently. Cotyledon removal has different effects on heterosis in different hybrids. After cotyledon removal, in C24xLer hybrids, both growth and heterosis were reduced in similar proportions, but the level of hybrid vigor was reduced as a proportion of growth in C24xColumbia (Col) and ColxLer hybrids. The removal of cotyledons at 4 DAS markedly decreased the level of growth and eliminated the heterotic phenotype of Wassilewskija (Ws)/Ler hybrids. In mutant Ws/Ler hybrids which had a reduced level of photosynthesis in the cotyledons, there was a reduction in plant growth and loss of heterosis. The variation in contribution of cotyledons to heterosis in different hybrids indicates there are multiple pathways to achieve heterotic phenotypes.

Early changes of gene activity in developing seedlings of Arabidopsis hybrids relative to parents may contribute to hybrid vigour.

Hybrid vigour (heterosis) has been used for decades in crop industries, especially in the production of maize and rice. Hybrid varieties usually exceed their parents in plant biomass and seed yield. But the molecular basis of hybrid vigour is not fully understood. In this project, we studied heterosis at early stages of seedling development in Arabidopsis hybrids derived from crossing Ler and C24 accessions. We found that early heterosis is associated with non-additive gene expression that resulted from earlier changes in gene expression in the hybrids relative to the parents. The non-additively expressed genes are involved in metabolic pathways, including photosynthesis, critical for plant growth. The early increased expression levels of genes involved in energy production in hybrids is associated with heterosis in the young seedlings that could be essential for biomass heterosis at later developmental stages of the plant.

Epigenetic Changes in Hybrids.

Genome-wide approaches to the study of hybrid vigor have identified epigenetic changes in the hybrid nucleus in Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and rice (Oryza sativa). DNA methylation associated with 24-nucleotide small interfering RNAs exhibits transallelic effects in hybrids of Arabidopsis and other species. Some of the transmethylation changes are inherited and some affect gene expression. Hybrids have larger leaves than those of the parents and have increases in cell size and number. The increased leaf size results in a greater photosynthetic capacity, which may support the increased vegetative and reproductive yields of the F1 hybrids. Genes and metabolic pathways that have altered expression relative to the parents include loci involved in responses to hormones and to biotic and abiotic stress. Whereas epigenetically induced changes in gene expression may contribute to hybrid vigor, the link between the transcriptional changes and the hybrid phenotype is not confirmed. Recurrent selection of high yielding F1 lines from the F2/F3 of a number of crops has fixed heterosis yields in pure breeding lines. These hybrid-like lines may have valuable applications in crop systems.