Basement membranes possess a complex three dimensional topography in the nanoscale and submicron range which have been shown to profoundly modulate a large menu of fundamental cell behaviors. Using the topographic features found in native vascular endothelial basement membranes as a guide, polyurethane substrates were fabricated containing anisotropically ordered ridge and groove structures and isotropically ordered pores from 200 nm to 2000 nm in size. We investigated the impact of biomimetic length-scale topographic cues on orientation/elongation, proliferation and migration on four human vascular endothelial cell-types from large and small diameter vessels. We found that all cell-types exhibited orientation and alignment with the most pronounced response on anisotropically ordered ridges ≥ 800 nm. HUVEC cells were the only cell-type examined to demonstrate a decrease in proliferation in response to the smallest topographic features regardless of surface order. On anisotropically ordered surfaces all cell types migrated preferentially parallel to the long axis of the ridges, with the greatest increase in cell migration being observed on the 1200 nm pitch. In contrast, cells did not exhibit any preference in direction or increase in migration speed on isotropically ordered surfaces. Overall, our data demonstrate that surface topographic features impact vascular endothelial cell behavior and that the impact of features varies with the cell behavior being considered, topographic feature scale, surface order, and the anatomic origin of the cell being investigated.

Recent studies have shown that nanoscale and submicron topographic cues modulate a menu of fundamental cell behaviors, and the use of topographic cues is an expanding area of study in tissue engineering. We used topographically-patterned substrates containing anisotropically-ordered ridges and grooves to investigate the effects of topographic cues on mesenchymal stem cell morphology, proliferation, and osteogenic differentiation. We found that human mesenchymal stem cells cultured on 1400 or 4000 nm pitches showed greater elongation and alignment relative to 400 nm pitch or planar control. Cells cultured on 400 nm pitch demonstrated significant increases in RUNX2 and BGLAP expression relative to cells cultured on 1400 or 4000 nm pitch or planar control. 400 nm pitch enhanced extracellular calcium deposition. Cells cultured in osteoinductive medium revealed combinatory effects of topography and chemical cues on 400 nm pitch as well as up-regulation of expression of ID1, a target of the BMP pathway. Our data demonstrate that a specific size scale of topographic cue promotes osteogenic differentiation with or without osteogenic agents. These data demonstrate that the integration of topographic cues may be useful for the fabrication of orthopedic implants.

The common ice plant (Mesembryanthemum crystallinum L.) has emerged as a useful model for molecular genetic studies of Crassulacean acid metabolism (CAM) because CAM can be induced in this species by water deficit or salinity stress. Non-redundant sequence information from expressed sequence tag data was used to fabricate a custom oligonucleotide microarray to compare large-scale mRNA expression patterns in M. crystallinum plants conducting C(3) photosynthesis versus CAM. Samples were collected every 4 h over a 24 h time period at the start of the subjective second day from plants grown under constant light and temperature conditions in order to capture variation in mRNA expression due to salinity stress and circadian clock control. Of 8455 genes, a total of 2343 genes (approximately 28%) showed a significant change as judged by analysis of variance (ANOVA) in steady-state mRNA abundance at one or more time points over the 24 h period. Of these, 858 (10%) and 599 (7%) exhibited a greater than two-fold ratio (TFR) increase or decrease in mRNA abundance, respectively. Functional categorization of these TFR genes revealed that many genes encoding products that function in CAM-related C(4) acid carboxylation/decarboxylation, glycolysis/gluconeogenesis, polysaccharide, polyol, and starch biosynthesis/degradation, protein degradation, transcriptional activation, signalling, stress response, and transport facilitation, and novel, unclassified proteins exhibited stress-induced increases in mRNA abundance. In contrast, salt stress resulted in a significant decrease in transcript abundance for genes encoding photosynthetic functions, protein synthesis, and cellular biogenesis functions. Many genes with CAM-related functions exhibited phase shifts in their putative circadian expression patterns following CAM induction. This report establishes an extensive catalogue of gene expression patterns for future investigations aimed at understanding the complex, transcriptional hierarchies that govern CAM-specific expression patterns. A novel graph-theoretic approach called 'Max Clique Builder' is introduced that identifies and organizes sets of coordinately regulated genes, such as those encoding subunits of the vacuolar H(+)-ATPase complex, into tighter functionally related clusters with more similar expression patterns compared with standard hierarchical clustering methods.

Glaucoma is a devastating neurodegenerative disease, which can lead to vision loss and is associated with irreversible damage to retinal ganglion cells. Although the mechanism of disease onset remains unknown, we have recently demonstrated that the stiffness of the ocular trabecular meshwork (HTM) increases dramatically in human donor eyes with a history of glaucoma. Here we report that polyacrylamide hydrogels, which mimic the compliant conditions of normal and glaucomatous HTM, profoundly modulate cytoskeletal dynamics and the elastic modulus of the overlying HTM cells. Substratum compliance also modulates HTM cell response to Latrunculin-B, a cytoskeletal disrupting agent currently in human clinical trials for the treatment of glaucoma. Additionally, we observed a compliance-dependent rebound effect of Latrunculin-B with an unexpected increase in HTM cell elastic modulus being observed upon withdrawal of the drug. The results predict that cytoskeletal disrupting drugs may be more potent in advanced stages of glaucoma.

The developmental time of period mutants in Drosophila melanogaster was monitored under different environmental conditions. We observed that the pe? mutants, which have short 19 h circadian cycles, develop faster from eggs to adult than the wild-type: perL mutants, which have long 28 h circadian rhythms, complete development more slowly than the wild-type. These results suggest that endogenous timers may be involved in regulating the development time of D. melanogaster.