Racioethnic differences in the biomechanical response of the lamina cribrosa.

Glaucoma is the second leading cause of irreversible blindness in the world with a higher prevalence in those of African Descent (AD) and Hispanic Ethnicity (HE) than in those of European Descent (ED). The objective of this study was to investigate the pressure dependent biomechanical response of the lamina cribrosa (LC) in normal human donor tissues from these racioethnic backgrounds. Pressure inflation tests were performed on 24 human LCs (n = 9 AD, n = 6 ED, and n = 9 HE) capturing the second harmonic generation (SHG) signal of collagen at 5, 15, 30, and 45 mmHg from an anterior view. A non-rigid image registration technique was utilized to determine the 3D displacement field in each LC from which 3D Green strains were calculated. The peak shear strain in the superior quadrant of the LC in those of ED was significantly higher than in those of AD and HE (p-value = 0.005 & 0.034, respectively) where ED = 0.017 [IQR = 0.012-0.027], AD = 0.0002 [IQR = -0.001-0.007], HE = 0.0016 [IQR = -0.002-0.012]). There were also significant differences in the regional strain heterogeneity in those of AD and HE that were absent in those of ED. This work represents, to our knowledge, the first ex-vivo study identifying significant differences in the biomechanical response of the LC in populations at increased risk of glaucoma. Future work will be necessary to assess if and how these differences play a role in predisposing those of Hispanic Ethnicity and African Descent to the onset and/or progression of primary open angle glaucoma. STATEMENT OF SIGNIFICANCE: Glaucoma is the second leading cause of irreversible blindness in the world and occurs more frequently in those of African Descent and Hispanic Ethnicity than in those of European Descent. To date, there has been no ex-vivo study quantifying differences in the biomechanical response of the non-glaucomatous lamina cribrosa (LC) across these racioethnic backgrounds. In this work we report, for the first time, differences in the pressure dependent biomechanical response of LC across different racioethnic groups as quantified using nonlinear optical microscopy. This study lays the foundation for future work investigating if and how these differences may play a role in predisposing those at increased risk to the onset and/or progression of primary open angle glaucoma.

Three-Dimensional Segmentation of the Ex-Vivo Anterior Lamina Cribrosa From Second-Harmonic Imaging Microscopy.

The lamina cribrosa (LC) is a connective tissue in the posterior eye with a complex mesh-like trabecular microstructure, through which all the retinal ganglion cell axons and central retinal vessels pass. Recent studies have demonstrated that changes in the structure of the LC correlate with glaucomatous damage. Thus, accurate segmentation and reconstruction of the LC is of utmost importance. This paper presents a new automated method for segmenting the microstructure of the anterior LC in the images obtained via multiphoton microscopy using a combination of ideas. In order to reduce noise, we first smooth the input image using a 4-D collaborative filtering scheme. Next, we enhance the beam-like trabecular microstructure of the LC using wavelet multiresolution analysis. The enhanced LC microstructure is then automatically extracted using a combination of histogram thresholding and graph-cut binarization. Finally, we use morphological area opening as a postprocessing step to remove the small and unconnected 3-D regions in the binarized images. The performance of the proposed method is evaluated using mutual overlap accuracy, Tanimoto index, F-score, and Rand index. Quantitative and qualitative results show that the proposed algorithm provides improved segmentation accuracy and computational efficiency compared to the other recent algorithms.

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.