TGFß2 differentially modulates smooth muscle cell proliferation and migration in electrospun gelatin-fibrinogen constructs.

A main goal of tissue engineering is the development of scaffolds that replace, restore and improve injured tissue. These scaffolds have to mimic natural tissue, constituted by an extracellular matrix (ECM) support, cells attached to the ECM, and signaling molecules such as growth factors that regulate cell function. In this study we created electrospun flat sheet scaffolds using different compositions of gelatin and fibrinogen. Smooth muscle cells (SMCs) were seeded on the scaffolds, and proliferation and infiltration were evaluated. Additionally, different concentrations of Transforming Growth Factor-beta2 (TGFß2) were added to the medium with the aim of elucidating its effect on cell proliferation, migration and collagen production. Our results demonstrated that a scaffold with a composition of 80% gelatin-20% fibrinogen is suitable for tissue engineering applications since it promotes cell growth and migration. The addition of TGFß2 at low concentrations (≤ 1 ng/ml) to the culture medium resulted in an increase in SMC proliferation and scaffold infiltration, and in the reduction of collagen production. In contrast, TGFß2 at concentrations >1 ng/ml inhibited cell proliferation and migration while stimulating collagen production. According to our results TGFß2 concentration has a differential effect on SMC function and thus can be used as a biochemical modulator that can be beneficial for tissue engineering applications.

Differences in the region- and depth-dependent microstructural organization in normal versus glaucomatous human posterior sclerae.

PURPOSE: This study quantitatively investigated differences in the regional- and depth-dependent human posterior scleral microstructure in glaucomatous (G) and nonglaucomatous (NG) donors. METHODS: Twenty-five posterior poles from six G and seven NG donors were analyzed using small angle light scattering (SALS) to investigate the organization of scleral fibers around the optic nerve head. Eccentricity (Ecc), fiber splay (FS), and percent equatorial fibers (PEF) were quantified. RESULTS: Regional statistically significant differences between G and NG groups existed in Ecc (P < 0.0001), FS (P < 0.005), and PEF (P < 0.005). Distinct and substantial variation through the depth occurred in all three end points. Region-specific differences in Ecc existed at the episcleral surface; however, by 40% into the depth, all regions converged to a similar value. Fiber splay increased in all regions by an average of 0.14 from the episcleral surface to the intraocular surface. The percentage of equatorial fibers decreased universally through the depth from approximately 61% to 33%. Generally, the inferior and superior regions had a lower Ecc and PEF compared to the nasal and temporal regions. CONCLUSIONS: Region and depth of the posterior sclera are important factors that should be included when comparing scleral microstructure of G and NG tissue in experimental and computational work. The dramatic changes in the depth of the sclera may represent baseline properties that affect predisposition to primary open angle glaucoma (POAG), and necessitate that further research include depth as a factor in assessing how observed structural differences contribute to or are a result of POAG.

Spatiotemporal analysis of copper homeostasis in Populus trichocarpa reveals an integrated molecular remodeling for a preferential allocation of copper to plastocyanin in the chloroplasts of developing leaves.

Plastocyanin, which requires copper (Cu) as a cofactor, is an electron carrier in the thylakoid lumen and essential for photoautotrophic growth of plants. The Cu microRNAs, which are expressed during Cu deprivation, down-regulate several transcripts that encode for Cu proteins. Since plastocyanin is not targeted by the Cu microRNAs, a cofactor economy model has been proposed in which plants prioritize Cu for use in photosynthetic electron transport. However, defects in photosynthesis are classic symptoms of Cu deprivation, and priorities in Cu cofactor delivery have not been determined experimentally. Using hydroponically grown Populus trichocarpa (clone Nisqually-1), we have established a physiological and molecular baseline for the response to Cu deficiency. An integrated analysis showed that Cu depletion strongly reduces the activity of several Cu proteins including plastocyanin, and consequently, photosynthesis and growth are decreased. Whereas plastocyanin mRNA levels were only mildly affected by Cu depletion, this treatment strongly affected the expression of other Cu proteins via Cu microRNA-mediated transcript down-regulation. Polyphenol oxidase was newly identified as Cu regulated and targeted by a novel Cu microRNA, miR1444. Importantly, a spatiotemporal analysis after Cu resupply to previously depleted plants revealed that this micronutrient is preferentially allocated to developing photosynthetic tissues. Plastocyanin and photosynthetic electron transport efficiency were the first to recover after Cu addition, whereas recovery of the other Cu-dependent activities was delayed. Our findings lend new support to the hypothesis that the Cu microRNAs serve to mediate a prioritization of Cu cofactor use. These studies also highlight poplar as an alternative sequenced model for spatiotemporal analyses of nutritional homeostasis.

Demonstration that drug-targeted down-regulation of MYC in non-Hodgkins lymphoma is directly mediated through the promoter G-quadruplex.

Most transcription of the MYC proto-oncogene initiates in the near upstream promoter, within which lies the nuclease hypersensitive element (NHE) III(1) region containing the CT-element. This dynamic stretch of DNA can form at least three different topologies: single-stranded DNA, double-stranded DNA, or higher order secondary structures that silence transcription. In the current report, we identify the ellipticine analog GQC-05 (NSC338258) as a high affinity, potent, and selective stabilizer of the MYC G-quadruplex (G4). In cells, GQC-05 induced cytotoxicity with corresponding decreased MYC mRNA and altered protein binding to the NHE III(1) region, in agreement with a G4 stabilizing compound. We further describe a unique feature of the Burkitt's lymphoma cell line CA46 that allowed us to clearly demonstrate the mechanism and location of action of GQC-05 within this region of DNA and through the G4. Most importantly, these data present, as far as we are aware, the most direct evidence of intracellular G4-mediated control of a particular promoter.