Identification of novel angiogenesis inhibitors

Vascular endothelial growth factor (VEGF) is a key stimulant of angiogenesis, which is the process of generating new capillary blood vessels. Inhibition of the vascular endothelial growth factor receptor (VEGFR) kinase is known to result in blockage of angiogenesis. A pharmacophore was developed based on the binding of ATP to the hinge region of the kinase domain of VEGFR and a database search of 18,000 compounds was conducted. Selected hits were assessed for their ability to limit the induction of web-like network of capillary tubes by the human umbilical vascular endothelial cells. Two compounds (1 and 4) showed good inhibitory ability to prevent sprouting and closed polygon formation of the tubular networks, promising them to be lead compounds. Compound 4 showed 60% inhibition at 0.05 microM.     

Recurrent heat shock impairs the proliferation and differentiation of C2C12 myoblasts

Heat-related illness and injury are becoming a growing safety concern for the farmers, construction workers, miners, firefighters, manufacturing workers, and other outdoor workforces who are exposed to heat stress in their routine lives. A primary response by a cell to an acute heat shock (HS) exposure is the induction of heat-shock proteins (HSPs), which chaperone and facilitate cellular protein folding and remodeling processes. While acute HS is well studied, the effect of repeated bouts of hyperthermia and the sustained production of HSPs in the myoblast-myotube model system of C2C12 cells are poorly characterized. In C2C12 myoblasts, we found that robust HS (43 °C, dose/time) significantly decreased the proliferation by 50% as early as on day 1 and maintained at the same level on days 2 and 3 of HS. This was accompanied by an accumulation of cells at G2 phase with reduced cell number in G1 phase indicating cell cycle arrest. FACS analysis indicates that there was no apparent change in apoptosis (markers) and cell death upon repeated HS. Immunoblot analysis and qPCR demonstrated a significant increase in the baseline expression of HSP25, 70, and 90 (among others) in cells after a single HS (43 °C) for 60 min as a typical HS response. Importantly, the repeated HS for 60 min each on days 2 and 3 maintained the elevated levels of HSPs compared to the control cells. Further, the continuous HS exposure resulted in significant inhibition of the differentiation of C2C12 myocytes to myotubes and only 1/10th of the cells underwent differentiation in HS relative to control. This was associated with significantly higher levels of HSPs and reduced expression of myogenin and Myh2 (P < 0.05), the genes involved in the differentiation process. Finally, the cell migration (scratch) assay indicated that the wound closure was significantly delayed in HS cells relative to the control cells. Overall, these results suggest that a repeated HS may perturb the active process of proliferation, motility, and differentiation processes in an in vitro murine myoblast-myotube model.     

Glaucomatous Patterns in Frequency Doubling Technology (FDT) Perimetry Data Identified by Unsupervised Machine Learning Classifiers

Purpose

The variational Bayesian independent component analysis-mixture model (VIM), an unsupervised machine-learning classifier, was used to automatically separate Matrix Frequency Doubling Technology (FDT) perimetry data into clusters of healthy and glaucomatous eyes, and to identify axes representing statistically independent patterns of defect in the glaucoma clusters.

Methods

FDT measurements were obtained from 1,190 eyes with normal FDT results and 786 eyes with abnormal FDT results from the UCSD-based Diagnostic Innovations in Glaucoma Study (DIGS) and African Descent and Glaucoma Evaluation Study (ADAGES). For all eyes, VIM input was 52 threshold test points from the 24-2 test pattern, plus age.

Results

FDT mean deviation was −1.00 dB (S.D. = 2.80 dB) and −5.57 dB (S.D. = 5.09 dB) in FDT-normal eyes and FDT-abnormal eyes, respectively (p<0.001). VIM identified meaningful clusters of FDT data and positioned a set of statistically independent axes through the mean of each cluster. The optimal VIM model separated the FDT fields into 3 clusters. Cluster N contained primarily normal fields (1109/1190, specificity 93.1%) and clusters G₁ and G₂ combined, contained primarily abnormal fields (651/786, sensitivity 82.8%). For clusters G₁ and G₂ the optimal number of axes were 2 and 5, respectively. Patterns automatically generated along axes within the glaucoma clusters were similar to those known to be indicative of glaucoma. Fields located farther from the normal mean on each glaucoma axis showed increasing field defect severity.

Conclusions

VIM successfully separated FDT fields from healthy and glaucoma eyes without a priori information about class membership, and identified familiar glaucomatous patterns of loss.     

The Effect of Activated Charcoal Supplemented Media to Browning of In Vitro Cultures of Piper species

With the aim to effectively minimise the browning of tissue cultures of different Piper species (P. longum, P. attenuatum, P. betle, P. nigrum) explants from stem (nodal and internodal segments), petiole, and leaf were planted on Murashige and Skoog's basal medium supplemented with activated charcoal (AC). AC in the concentration of 200 mg dm^-3 proved to be the best. Among the taxa studied, P. longum showed the least browning whereas, wild P. nigrum showed maximum browning.     

Effect of colchicine induced tetraploidy on morphology,cytology, essential oil composition,gene expression and antioxidant activity of Citrus limon (L.) Osbeck

Citrus limon (L.) Osbeck cultivated all over the world is a valuable source of aromatic essential oil. To develop tetraploids of C. limon, four different concentration of colchicine (0.025, 0.05, 0.1 and 0.2%; w/v) and three varied exposure time (12, 24 and 48 h) were employed. The ploidy level of diploids (2n = 2x = 18) and tetraploids (2n = 4x = 36) were determined by direct chromosome count and confirmed by flow cytometric analyses. Successful result with maximum tetraploidy frequency was observed in plantlets developed from seeds treated with 0.025% colchicine for 24 h. Morphological and stomatal characteristics indicated that tetraploids were taller with increased leaf and root length. On the other side, the leaves of tetraploids had fewer and larger stomata with a greater number of chloroplasts in guard cells in contrast with diploids. GC–GC/MS analyses showed cyclic monoterpene, limonene had increased significantly in tetraploids and was further confirmed by HPLC quantification. RT-PCR analyses revealed unaltered expression of monoterpene synthase, sesquiterpene synthase and flavone synthase and remarkable upregulation of genes such as limonene synthase, chalcone synthase and phenylalanine ammonia lyase in tetraploids. Antioxidant activity of essential oil of tetraploids was higher than diploids in all the five test systems studied. Overall, the findings of the present study prove that colchicine induced tetraploidy in C. limon could be a greater source of essential oil with improved composition and of economic significance.